Variants in ZFX are associated with an X-linked neurodevelopmental disorder with recurrent facial gestalt.

Pathogenic variants in multiple genes on the X chromosome have been implicated in syndromic and non-syndromic intellectual disability disorders. ZFX on Xp22.11 encodes a transcription factor that has been linked to diverse processes including oncogenesis and development, but germline variants have not been characterized in association with disease. Here, we present clinical and molecular characterization of 18 individuals with germline ZFX variants. Exome or genome sequencing revealed 11 variants in 18 subjects (14 males and 4 females) from 16 unrelated families. Four missense variants were identified in 11 subjects, with seven truncation variants in the remaining individuals. Clinical findings included developmental delay/intellectual disability, behavioral abnormalities, hypotonia, and congenital anomalies. Overlapping and recurrent facial features were identified in all subjects, including thickening and medial broadening of eyebrows, variations in the shape of the face, external eye abnormalities, smooth and/or long philtrum, and ear abnormalities. Hyperparathyroidism was found in four families with missense variants, and enrichment of different tumor types was observed. In molecular studies, DNA-binding domain variants elicited differential expression of a small set of target genes relative to wild-type ZFX in cultured cells, suggesting a gain or loss of transcriptional activity. Additionally, a zebrafish model of ZFX loss displayed an altered behavioral phenotype, providing additional evidence for the functional significance of ZFX. Our clinical and experimental data support that variants in ZFX are associated with an X-linked intellectual disability syndrome characterized by a recurrent facial gestalt, neurocognitive and behavioral abnormalities, and an increased risk for congenital anomalies and hyperparathyroidism.

Bi-allelic variants in IPO8 cause a connective tissue disorder associated with cardiovascular defects, skeletal abnormalities, and immune dysregulation.

Dysregulated transforming growth factor TGF-ß signaling underlies the pathogenesis of genetic disorders affecting the connective tissue such as Loeys-Dietz syndrome. Here, we report 12 individuals with bi-allelic loss-of-function variants in IPO8 who presented with a syndromic association characterized by cardio-vascular anomalies, joint hyperlaxity, and various degree of dysmorphic features and developmental delay as well as immune dysregulation; the individuals were from nine unrelated families. Importin 8 belongs to the karyopherin family of nuclear transport receptors and was previously shown to mediate TGF-ß-dependent SMADs trafficking to the nucleus in vitro. The important in vivo role of IPO8 in pSMAD nuclear translocation was demonstrated by CRISPR/Cas9-mediated inactivation in zebrafish. Consistent with IPO8's role in BMP/TGF-ß signaling, ipo8-/- zebrafish presented mild to severe dorso-ventral patterning defects during early embryonic development. Moreover, ipo8-/- zebrafish displayed severe cardiovascular and skeletal defects that mirrored the human phenotype. Our work thus provides evidence that IPO8 plays a critical and non-redundant role in TGF-ß signaling during development and reinforces the existing link between TGF-ß signaling and connective tissue defects.

Narrowing the diagnostic gap: Genomes, episignatures, long-read sequencing, and health economic analyses in an exome-negative intellectual disability cohort.

PURPOSE: Genome sequencing (GS)-specific diagnostic rates in prospective tightly ascertained exome sequencing (ES)-negative intellectual disability (ID) cohorts have not been reported extensively. METHODS: ES, GS, epigenetic signatures, and long-read sequencing diagnoses were assessed in 74 trios with at least moderate ID. RESULTS: The ES diagnostic yield was 42 of 74 (57%). GS diagnoses were made in 9 of 32 (28%) ES-unresolved families. Repeated ES with a contemporary pipeline on the GS-diagnosed families identified 8 of 9 single-nucleotide variations/copy-number variations undetected in older ES, confirming a GS-unique diagnostic rate of 1 in 32 (3%). Episignatures contributed diagnostic information in 9% with GS corroboration in 1 of 32 (3%) and diagnostic clues in 2 of 32 (6%). A genetic etiology for ID was detected in 51 of 74 (69%) families. Twelve candidate disease genes were identified. Contemporary ES followed by GS cost US$4976 (95% CI: $3704; $6969) per diagnosis and first-line GS at a cost of $7062 (95% CI: $6210; $8475) per diagnosis. CONCLUSION: Performing GS only in ID trios would be cost equivalent to ES if GS were available at $2435, about a 60% reduction from current prices. This study demonstrates that first-line GS achieves higher diagnostic rate than contemporary ES but at a higher cost.

Klinefelter Syndrome: What should we tell prospective parents?

Klinefelter syndrome (KS) or 47,XXY is the most common sex chromosome aneuploidy (SCA), occurring at a prevalence of 1 in 600 male pregnancies. Historically, only 25% of individuals with KS came to medical attention, for a range of issues across the life course including under-virilisation at birth, developmental and social concerns in childhood, absence, delay or arrest of puberty in adolescence or infertility in adulthood. Our understanding of the phenotypic spectrum of KS has been largely influenced by this ascertainment bias. With increasing uptake of antenatal noninvasive prenatal testing (NIPT), a corresponding increase in identification of KS has been documented. Population-based longitudinal data from infancy to adulthood on these individuals is lacking, which impedes balanced antenatal genetic counselling and raises issues for prospective parents and clinicians alike.

Lessons learnt from multifaceted diagnostic approaches to the first 150 families in Victoria's Undiagnosed Diseases Program.

BACKGROUND: Clinical exome sequencing typically achieves diagnostic yields of 30%-57.5% in individuals with monogenic rare diseases. Undiagnosed diseases programmes implement strategies to improve diagnostic outcomes for these individuals. AIM: We share the lessons learnt from the first 3 years of the Undiagnosed Diseases Program-Victoria, an Australian programme embedded within a clinical genetics service in the state of Victoria with a focus on paediatric rare diseases. METHODS: We enrolled families who remained without a diagnosis after clinical genomic (panel, exome or genome) sequencing between 2016 and 2018. We used family-based exome sequencing (family ES), family-based genome sequencing (family GS), RNA sequencing (RNA-seq) and high-resolution chromosomal microarray (CMA) with research-based analysis. RESULTS: In 150 families, we achieved a diagnosis or strong candidate in 64 (42.7%) (37 in known genes with a consistent phenotype, 3 in known genes with a novel phenotype and 24 in novel disease genes). Fifty-four diagnoses or strong candidates were made by family ES, six by family GS with RNA-seq, two by high-resolution CMA and two by data reanalysis. CONCLUSION: We share our lessons learnt from the programme. Flexible implementation of multiple strategies allowed for scalability and response to the availability of new technologies. Broad implementation of family ES with research-based analysis showed promising yields post a negative clinical singleton ES. RNA-seq offered multiple benefits in family ES-negative populations. International data sharing strategies were critical in facilitating collaborations to establish novel disease-gene associations. Finally, the integrated approach of a multiskilled, multidisciplinary team was fundamental to having diverse perspectives and strategic decision-making.

Mosaicism in tuberous sclerosis complex: Lowering the threshold for clinical reporting.

Tuberous sclerosis complex (TSC) is a multi-system genetic disorder. Most patients have germline mutations in TSC1 or TSC2 but, 10%-15% patients do not have TSC1/TSC2 mutations detected on routine clinical genetic testing. We investigated the contribution of low-level mosaic TSC1/TSC2 mutations in unsolved sporadic patients and families with TSC. Thirty-one sporadic TSC patients negative on routine testing and eight families with suspected parental mosaicism were sequenced using deep panel sequencing followed by droplet digital polymerase chain reaction. Pathogenic variants were found in 22/31 (71%) unsolved sporadic patients, 16 were mosaic (median variant allele fraction [VAF] 6.8% in blood) and 6 had missed germline mutations. Parental mosaicism was detected in 5/8 families (median VAF 1% in blood). Clinical testing laboratories typically only report pathogenic variants with allele fractions above 10%. Our findings highlight the critical need to change laboratory practice by implementing higher sensitivity assays to improve diagnostic yield, inform patient management and guide reproductive counseling.

Genomic and phenotypic characterization of 404 individuals with neurodevelopmental disorders caused by CTNNB1 variants.

PURPOSE: Germline loss-of-function variants in CTNNB1 cause neurodevelopmental disorder with spastic diplegia and visual defects (NEDSDV; OMIM 615075) and are the most frequent, recurrent monogenic cause of cerebral palsy (CP). We investigated the range of clinical phenotypes owing to disruptions of CTNNB1 to determine the association between NEDSDV and CP. METHODS: Genetic information from 404 individuals with collectively 392 pathogenic CTNNB1 variants were ascertained for the study. From these, detailed phenotypes for 52 previously unpublished individuals were collected and combined with 68 previously published individuals with comparable clinical information. The functional effects of selected CTNNB1 missense variants were assessed using TOPFlash assay. RESULTS: The phenotypes associated with pathogenic CTNNB1 variants were similar. A diagnosis of CP was not significantly associated with any set of traits that defined a specific phenotypic subgroup, indicating that CP is not additional to NEDSDV. Two CTNNB1 missense variants were dominant negative regulators of WNT signaling, highlighting the utility of the TOPFlash assay to functionally assess variants. CONCLUSION: NEDSDV is a clinically homogeneous disorder irrespective of initial clinical diagnoses, including CP, or entry points for genetic testing.

Neurogenetic fetal akinesia and arthrogryposis: genetics, expanding genotype-phenotypes and functional genomics.

BACKGROUND: Fetal akinesia and arthrogryposis are clinically and genetically heterogeneous and have traditionally been refractive to genetic diagnosis. The widespread availability of affordable genome-wide sequencing has facilitated accurate genetic diagnosis and gene discovery in these conditions. METHODS: We performed next generation sequencing (NGS) in 190 probands with a diagnosis of arthrogryposis multiplex congenita, distal arthrogryposis, fetal akinesia deformation sequence or multiple pterygium syndrome. This sequencing was a combination of bespoke neurogenetic disease gene panels and whole exome sequencing. Only class 4 and 5 variants were reported, except for two cases where the identified variants of unknown significance (VUS) are most likely to be causative for the observed phenotype. Co-segregation studies and confirmation of variants identified by NGS were performed where possible. Functional genomics was performed as required. RESULTS: Of the 190 probands, 81 received an accurate genetic diagnosis. All except two of these cases harboured class 4 and/or 5 variants based on the American College of Medical Genetics and Genomics guidelines. We identified phenotypic expansions associated with CACNA1S, CHRNB1, GMPPB and STAC3. We describe a total of 50 novel variants, including a novel missense variant in the recently identified gene for arthrogryposis with brain malformations-SMPD4. CONCLUSIONS: Comprehensive gene panels give a diagnosis for a substantial proportion (42%) of fetal akinesia and arthrogryposis cases, even in an unselected cohort. Recently identified genes account for a relatively large proportion, 32%, of the diagnoses. Diagnostic-research collaboration was critical to the diagnosis and variant interpretation in many cases, facilitated genotype-phenotype expansions and reclassified VUS through functional genomics.

Biallelic hypomorphic variants in ALDH1A2 cause a novel lethal human multiple congenital anomaly syndrome encompassing diaphragmatic, pulmonary, and cardiovascular defects.

This study shows a causal association between ALDH1A2 variants and a novel, severe multiple congenital anomaly syndrome in humans that is neonatally lethal due to associated pulmonary hypoplasia and respiratory failure. In two families, exome sequencing identified compound heterozygous missense variants in ALDH1A2. ALDH1A2 is involved in the conversion of retinol (vitamin A) into retinoic acid (RA), which is an essential regulator of diaphragm and cardiovascular formation during embryogenesis. Reduced RA causes cardiovascular, diaphragmatic, and associated pulmonary defects in several animal models, matching the phenotype observed in our patients. In silico protein modeling showed probable impairment of ALDH1A2 for three of the four substitutions. In vitro studies show a reduction of RA. Few pathogenic variants in genes encoding components of the retinoic signaling pathway have been described to date, likely due to embryonic lethality. Thus, this study contributes significantly to knowledge of the role of this pathway in human diaphragm and cardiovascular development and disease. Some clinical features in our patients are also observed in Fryns syndrome (MIM# 229850), syndromic microphthalmia 9 (MIM# 601186), and DiGeorge syndrome (MIM# 188400). Patients with similar clinical features who are genetically undiagnosed should be tested for recessive ALDH1A2-deficient malformation syndrome.

Increased nuchal translucency after low-risk noninvasive prenatal testing: What should we tell prospective parents?

Three decades ago, the observation that first trimester fetuses with excess fluid accumulation at the back of the neck were more likely to be aneuploid, gave rise to a new era of prenatal screening. The nuchal translucency (NT) measurement in combination with serum biomarkers and maternal age, resulted in the first trimester combined screening (FTCS) program. The introduction of noninvasive prenatal testing (NIPT) over the past decade has introduced the option for parents to receive highly sensitive and specific screening information for common trisomy from as early as 10 weeks gestation, altering the traditional pathway FTCS pathway. The retention of the 11-13-week NT ultrasound remains important in the detection of structural anomalies; however, the optimal management of pregnancies with a low-risk NIPT result and an isolated increased NT measurement in an era of advanced genomic testing options is a new dilemma for clinicians. For parents, the prolonged period between the initial diagnosis in first trimester, and prognostic information at each successive stage of investigations up to 22-24 weeks, can be emotionally challenging. This article addresses the common questions from parents and clinicians as they navigate the uncertainty of having a fetus diagnosed with an increased NT after a low-risk NIPT result and presents suggested approaches to management.

A cost-effectiveness analysis of genomic sequencing in a prospective versus historical cohort of complex pediatric patients.

PURPOSE: Cost-effectiveness evaluations of first-line genomic sequencing (GS) in the diagnosis of children with genetic conditions are limited by the lack of well-defined comparative cohorts. We sought to evaluate the cost-effectiveness of early GS in pediatric patients with complex monogenic conditions compared with a matched historical cohort. METHODS: Data, including investigation costs, were collected in a prospective cohort of 92 pediatric patients undergoing singleton GS over an 18-month period (2016-2017) with two of the following: a condition with high mortality, multisystem disease involving three or more organs, or severe limitation of daily function. Comparative data were collected in a matched historical cohort who underwent traditional investigations in the years 2012-2013. RESULTS: GS yielded a diagnosis in 42% while traditional investigations yielded a diagnosis in 23% (p = 0.003). A change in management was experienced by 74% of patients diagnosed following GS, compared with 32% diagnosed following traditional investigations. Singleton GS at a cost of AU$3100 resulted in a mean saving per person of AU$3602 (95% confidence interval [CI] AU$2520-4685). Cost savings occurred across all investigation subtypes and were only minimally offset by clinical management costs. CONCLUSION: GS in complex pediatric patients saves significant costs and doubles the diagnostic yield of traditional approaches.

Novel mutations in NLGN3 causing autism spectrum disorder and cognitive impairment.

The X-linked NLGN3 gene, encoding a postsynaptic cell adhesion molecule, was involved in a nonsyndromic monogenic form of autism spectrum disorder (ASD) by the description of one unique missense variant, p.Arg451Cys (Jamain et al. 2003). We investigated here the pathogenicity of additional missense variants identified in two multiplex families with intellectual disability (ID) and ASD: c.1789C>T, p.Arg597Trp, previously reported by our group (Redin et al. 2014) and present in three affected cousins and c.1540C>T, p.Pro514Ser, identified in two affected brothers. Overexpression experiments in HEK293 and HeLa cell lines revealed that both variants affect the level of the mature NLGN3 protein, its localization at the plasma membrane and its presence as a cleaved form in the extracellular environment, even more drastically than what was reported for the initial p.Arg451Cys mutation. The variants also induced an unfolded protein response, probably due to the retention of immature NLGN3 proteins in the endoplasmic reticulum. In comparison, the c.1894A>G, p.Ala632Thr and c.1022T>C, p.Val341Ala variants, present in males from the general population, have no effect. Our report of two missense variants affecting the normal localization of NLGN3 in a total of five affected individuals reinforces the involvement of the NLGN3 gene in a neurodevelopmental disorder characterized by ID and ASD.

EML1-associated brain overgrowth syndrome with ribbon-like heterotopia.

EML1 encodes the protein Echinoderm microtubule-associated protein-like 1 or EMAP-1 that binds to the microtubule complex. Mutations in this gene resulting in complex brain malformations have only recently been published with limited clinical descriptions. We provide further clinical and imaging details on three previously published families, and describe two novel unrelated individuals with a homozygous partial EML1 deletion and a homozygous missense variant c.760G>A, p.(Val254Met), respectively. From review of the clinical and imaging data of eight individuals from five families with biallelic EML1 variants, a very consistent imaging phenotype emerges. The clinical syndrome is characterized by mainly neurological features including severe developmental delay, drug-resistant seizures and visual impairment. On brain imaging there is megalencephaly with a characteristic ribbon-like subcortical heterotopia combined with partial or complete callosal agenesis and an overlying polymicrogyria-like cortical malformation. Several of its features can be recognized on prenatal imaging especially the abnormaly formed lateral ventricles, hydrocephalus (in half of the cases) and suspicion of a neuronal migration disorder. In conclusion, biallelic EML1 disease-causing variants cause a highly specific pattern of congenital brain malformations, severe developmental delay, seizures and visual impairment.

Genetic abnormalities in a large cohort of Coffin-Siris syndrome patients.

Coffin-Siris syndrome (CSS, MIM#135900) is a congenital disorder characterized by coarse facial features, intellectual disability, and hypoplasia of the fifth digit and nails. Pathogenic variants for CSS have been found in genes encoding proteins in the BAF (BRG1-associated factor) chromatin-remodeling complex. To date, more than 150 CSS patients with pathogenic variants in nine BAF-related genes have been reported. We previously reported 71 patients of whom 39 had pathogenic variants. Since then, we have recruited an additional 182 CSS-suspected patients. We performed comprehensive genetic analysis on these 182 patients and on the previously unresolved 32 patients, targeting pathogenic single nucleotide variants, short insertions/deletions and copy number variations (CNVs). We confirmed 78 pathogenic variations in 78 patients. Pathogenic variations in ARID1B, SMARCB1, SMARCA4, ARID1A, SOX11, SMARCE1, and PHF6 were identified in 48, 8, 7, 6, 4, 1, and 1 patients, respectively. In addition, we found three CNVs including SMARCA2. Of particular note, we found a partial deletion of SMARCB1 in one CSS patient and we thoroughly investigated the resulting abnormal transcripts.

Differential regulation of two FLNA transcripts explains some of the phenotypic heterogeneity in the loss-of-function filaminopathies.

Loss-of-function mutations in the X-linked gene FLNA can lead to abnormal neuronal migration, vascular and cardiac defects, and congenital intestinal pseudo-obstruction (CIPO), the latter characterized by anomalous intestinal smooth muscle layering. Survival in male hemizygotes for such mutations is dependent on retention of residual FLNA function but it is unclear why a subgroup of males with mutations in the 5' end of the gene can present with CIPO alone. Here, we demonstrate evidence for the presence of two FLNA isoforms differing by 28 residues at the N-terminus initiated at ATG+1 and ATG+82 . A male with CIPO (c.18_19del) exclusively expressed FLNA ATG+82 , implicating the longer protein isoform (ATG+1 ) in smooth muscle development. In contrast, mutations leading to reduction of both isoforms are associated with compound phenotypes affecting the brain, heart, and intestine. RNA-seq data revealed three distinct transcription start sites, two of which produce a protein isoform utilizing ATG+1 while the third utilizes ATG+82 . Transcripts sponsoring translational initiation at ATG+1 predominate in intestinal smooth muscle, and are more abundant compared with the level measured in fibroblasts. Together these observations describe a new mechanism of tissue-specific regulation of FLNA that could reflect the differing mechanical requirements of these cell types during development.

A mutation update on the LDS-associated genes TGFB2/3 and SMAD2/3.

The Loeys-Dietz syndrome (LDS) is a connective tissue disorder affecting the cardiovascular, skeletal, and ocular system. Most typically, LDS patients present with aortic aneurysms and arterial tortuosity, hypertelorism, and bifid/broad uvula or cleft palate. Initially, mutations in transforming growth factor-ß (TGF-ß) receptors (TGFBR1 and TGFBR2) were described to cause LDS, hereby leading to impaired TGF-ß signaling. More recently, TGF-ß ligands, TGFB2 and TGFB3, as well as intracellular downstream effectors of the TGF-ß pathway, SMAD2 and SMAD3, were shown to be involved in LDS. This emphasizes the role of disturbed TGF-ß signaling in LDS pathogenesis. Since most literature so far has focused on TGFBR1/2, we provide a comprehensive review on the known and some novel TGFB2/3 and SMAD2/3 mutations. For TGFB2 and SMAD3, the clinical manifestations, both of the patients previously described in the literature and our newly reported patients, are summarized in detail. This clearly indicates that LDS concerns a disorder with a broad phenotypical spectrum that is still emerging as more patients will be identified. All mutations described here are present in the corresponding Leiden Open Variant Database.

Monoallelic and biallelic CREB3L1 variant causes mild and severe osteogenesis imperfecta, respectively.

PurposeOsteogenesis imperfecta (OI) is a heritable skeletal dysplasia. Dominant pathogenic variants in COL1A1 and COL1A2 explain the majority of OI cases. At least 15 additional genes have been identified, but those still do not account for all OI phenotypes that present. We sought the genetic cause of mild and lethal OI phenotypes in an unsolved family.MethodsWe performed exome sequencing on seven members of the family, both affected and unaffected.ResultsWe identified a variant in cyclic AMP responsive element binding protein 3-like 1 (CREB3L1) in a consanguineous family. The variant caused a prenatal/perinatal lethal OI in homozygotes, similar to that seen in OI type II as a result of mutations in type I collagen genes, and a mild phenotype (fractures, blue sclerae) in multiple heterozygous family members. CREB3L1 encodes old astrocyte specifically induced substance (OASIS), an endoplasmic reticulum stress transducer. The variant disrupts a DNA-binding site and prevents OASIS from acting on its transcriptional targets including SEC24D, which encodes a component of the coat protein II complex.ConclusionThis report confirms that CREB3L1 is an OI-related gene and suggests the pathogenic mechanism of CREB3L1-associated OI involves the altered regulation of proteins involved in cellular secretion.

ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism.

Although mitochondrial disorders are clinically heterogeneous, they frequently involve the central nervous system and are among the most common neurogenetic disorders. Identifying the causal genes has benefited enormously from advances in high-throughput sequencing technologies; however, once the defect is known, researchers face the challenge of deciphering the underlying disease mechanism. Here we characterize large biallelic deletions in the region encoding the ATAD3C, ATAD3B and ATAD3A genes. Although high homology complicates genomic analysis of the ATAD3 defects, they can be identified by targeted analysis of standard single nucleotide polymorphism array and whole exome sequencing data. We report deletions that generate chimeric ATAD3B/ATAD3A fusion genes in individuals from four unrelated families with fatal congenital pontocerebellar hypoplasia, whereas a case with genomic rearrangements affecting the ATAD3C/ATAD3B genes on one allele and ATAD3B/ATAD3A genes on the other displays later-onset encephalopathy with cerebellar atrophy, ataxia and dystonia. Fibroblasts from affected individuals display mitochondrial DNA abnormalities, associated with multiple indicators of altered cholesterol metabolism. Moreover, drug-induced perturbations of cholesterol homeostasis cause mitochondrial DNA disorganization in control cells, while mitochondrial DNA aggregation in the genetic cholesterol trafficking disorder Niemann-Pick type C disease further corroborates the interdependence of mitochondrial DNA organization and cholesterol. These data demonstrate the integration of mitochondria in cellular cholesterol homeostasis, in which ATAD3 plays a critical role. The dual problem of perturbed cholesterol metabolism and mitochondrial dysfunction could be widespread in neurological and neurodegenerative diseases.

Contemporary prenatal aneuploidy screening practice in Australia: Frequently asked questions in the cell-free DNA era.

Cell-free DNA screening has quickly become established in Australia as an accurate - albeit costly - prenatal screening test for trisomy 21, 18 and 13. It is also commonly used for the detection of sex chromosome abnormalities. The increasing number of prenatal screening pathways available to women has increased the complexity of pretest counselling. Concurrent advances in diagnostic testing with the widespread use of chromosomal microarrays create further challenges for the continuing education of clinicians and health consumers. This article aims to answer common clinical questions in this rapidly evolving field and complements the recently updated Royal Australian and New Zealand College of Obstetricians and Gynaecologists Statement on Prenatal Screening for Fetal Chromosome and Genetic Conditions.