DISP1 deficiency: monoallelic and biallelic variants cause a spectrum of midline craniofacial malformations.

PURPOSE: DISP1 encodes a transmembrane protein that regulates the secretion of the morphogen, Sonic hedgehog (SHH), a deficiency of which is a major cause of holoprosencephaly (HPE). This disorder covers a spectrum of brain and midline craniofacial malformations. The objective of the present study was to better delineate the clinical phenotypes associated with DISP1 variants. METHODS: This study was based on the identification of at least one pathogenic variant of the DISP1 gene in individuals for whom detailed clinical data were available. RESULTS: A total of 23 DISP1 variants were identified in heterozygous, compound heterozygous or homozygous states in 25 individuals with midline craniofacial defects. Most cases were minor forms of HPE, with craniofacial features such as orofacial cleft, solitary median maxillary central incisor (SMMCI), and congenital nasal pyriform aperture stenosis (CNPAS). These individuals had either monoallelic loss-of-function variants or biallelic missense variants in DISP1. In individuals with severe HPE, the DISP1 variants were commonly found associated with a variant in another HPE-linked gene (i.e. oligogenic inheritance). CONCLUSION: The genetic findings we have acquired demonstrate a significant involvement of DISP1 variants in the phenotypic spectrum of midline defects. This underlines its importance as a crucial element in the efficient secretion of SHH. We also demonstrated that the very rare SMMCI-CNPAS combination is part of the DISP1-related phenotype. The present study highlights the clinical risks to be flagged up during genetic counseling after the discovery of a pathogenic DISP1 variant.

Molecular and Phenotypic Characterization of the RORB-Related Disorder.

BACKGROUND AND OBJECTIVES: Heterozygous variants in RAR-related orphan receptor B (RORB) have recently been associated with susceptibility to idiopathic generalized epilepsy. However, few reports have been published so far describing pathogenic variants of this gene in patients with epilepsy and intellectual disability (ID). In this study, we aimed to delineate the epilepsy phenotype associated with RORB pathogenic variants and to provide arguments in favor of the pathogenicity of variants. METHODS: Through an international collaboration, we analyzed seizure characteristics, EEG data, and genotypes of a cohort of patients with heterozygous variants in RORB. To gain insight into disease mechanisms, we performed ex vivo cortical electroporation in mouse embryos of 5 selected variants, 2 truncating and 3 missense, and evaluated on expression and quantified changes in axonal morphology. RESULTS: We identified 35 patients (17 male, median age 10 years, range 2.5-23 years) carrying 32 different heterozygous variants in RORB, including 28 single-nucleotide variants or small insertions/deletions (12 missense, 12 frameshift or nonsense, 2 splice-site variants, and 2 in-frame deletions), and 4 microdeletions; de novo in 18 patients and inherited in 10. Seizures were reported in 31/35 (89%) patients, with a median age at onset of 3 years (range 4 months-12 years). Absence seizures occurred in 25 patients with epilepsy (81%). Nineteen patients experienced a single seizure type: absences, myoclonic absences, or absences with eyelid myoclonia and focal seizures. Nine patients had absence seizures combined with other generalized seizure types. One patient had presented with absences associated with photosensitive occipital seizures. Three other patients had generalized tonic-clonic seizures without absences. ID of variable degree was observed in 85% of the patients. Expression studies in cultured neurons showed shorter axons for the 5 tested variants, both truncating and missense variants, supporting an impaired protein function. DISCUSSION: In most patients, the phenotype of the RORB-related disorder associates absence seizures with mild-to-moderate ID. In silico and in vitro evaluation of the variants in our cohort, including axonal morphogenetic experiments in cultured neurons, supports their pathogenicity, showing a hypomorphic effect.

Functional effects of disease-associated variants reveal that the S1-M1 linker of the NMDA receptor critically controls channel opening.

The short pre-M1 helix within the S1-M1 linker (also referred to as the pre-M1 linker) between the agonist-binding domain (ABD, S1) and the M1 transmembrane helix of the NMDA receptor (NMDAR) is devoid of missense variants within the healthy population but is a locus for de novo pathogenic variants associated with neurological disorders. Several de novo variants within this helix have been identified in patients presenting early in life with intellectual disability, developmental delay, and/or epilepsy. In this study, we evaluated functional properties for twenty variants within the pre-M1 linker in GRIN1, GRIN2A, and GRIN2B genes, including six novel missense variants. The effects of pre-M1 variants on agonist potency, sensitivity to endogenous allosteric modulators, response time course, channel open probability, and surface expression were assessed. Our data indicated that virtually all of the variants evaluated altered channel function, and multiple variants had profound functional consequences, which may contribute to the neurological conditions in the patients harboring the variants in this region. These data strongly suggest that the residues within the pre-M1 helix play a key role in channel gating and are highly intolerant to genetic variation.

Sudden Cardiac Arrest During a Sedated Cardiac Magnetic Resonance Study in a Nonsyndromic Child with Evolving Supravalvar Aortic Stenosis Due to Familial ELN Mutation.

Supravalvar aortic stenosis (SVAS) is a less common but clinically important form of left ventricular outflow tract obstruction, and commonly associated with Williams syndrome (WS). SVAS outside of WS may also occur sporadically or in a familial form, often with identifiable mutations in the elastin (ELN) gene. While risk of sudden cardiac death in patients with SVAS has been extensively described in the context of WS, less is known about risk in patients with isolated SVAS. We report a case of a nonsyndromic two-year-old boy with evolving manifestations of SVAS who developed sudden cardiac arrest and death during a sedated cardiac magnetic resonance imaging study. A strong family history of SVAS was present and targeted genetic testing identified an ELN gene mutation in the boy's affected father and other paternal relatives. We review risk factors found in the literature for SCA in SVAS patients and utilize this case to raise awareness of the risk of cardiac events in these individuals even in the absence of WS or severe disease. This case also underscores the importance of genetic testing, including targeted panels specifically looking for ELN gene mutations, in all patients with SVAS even in the absence of phenotypic concerns for WS or other genetic syndromes.

Familial Bainbridge-Ropers syndrome: Report of familial ASXL3 inheritance and a milder phenotype.

De novo truncating and splicing pathogenic variants in the Additional Sex Combs-Like 3 (ASXL3) gene are known to cause neurodevelopmental delay, intellectual disability, behavioral difficulties, hypotonia, feeding problems and characteristic facial features. We previously reported 45 patients with ASXL3-related disorder including three individuals with a familial variant. Here we report the detailed clinical and molecular characteristics of these three families with inherited ASXL3-related disorder. First, a father and son with c.2791_2792del p.Gln931fs pathogenic variant. The second, a mother, daughter and son with c.4534C > T, p.Gln1512Ter pathogenic variant. The third, a mother and her daughter with c.4441dup, p.Leu1481fs maternally inherited pathogenic variant. This report demonstrates intrafamilial phenotypic heterogeneity and confirms heritability of ASXL3-related disorder.

The clinical spectrum of SMA-PME and in vitro normalization of its cellular ceramide profile.

OBJECTIVE: The objectives of this study were to define the clinical and biochemical spectrum of spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) and to determine if aberrant cellular ceramide accumulation could be normalized by enzyme replacement. METHODS: Clinical features of 6 patients with SMA-PME were assessed by retrospective chart review, and a literature review of 24 previously published cases was performed. Leukocyte enzyme activity of acid ceramidase was assessed with a fluorescence-based assay. Skin fibroblast ceramide content and was assessed by high performance liquid chromatography, electrospray ionization tandem mass spectroscopy. Enzyme replacement was assessed using recombinant human acid ceramidase (rhAC) in vitro. RESULTS: The six new patients showed the hallmark features of SMA-PME, with variable initial symptom and age of onset. Five of six patients carried at least one of the recurrent SMA-PME variants observed in two specific codons of ASAH1. A review of 30 total cases revealed that patients who were homozygous for the most common c.125C > T variant presented in the first decade of life with limb-girdle weakness as the initial symptom. Sensorineural hearing loss was associated with the c.456A > C variant. Leukocyte acid ceramidase activity varied from 4.1%-13.1% of controls. Ceramide species in fibroblasts were detected and total cellular ceramide content was elevated by 2 to 9-fold compared to controls. Treatment with rhAC normalized ceramide profiles in cultured fibroblasts to control levels within 48 h. INTERPRETATION: This study details the genotype-phenotype correlations observed in SMA-PME and shows the impact of rhAC to correct the abnormal cellular ceramide profile in cells.

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.

Congenital hyperinsulinism in a newborn presenting with poor feeding.

Hyperinsulinemic hypoglycemia is a condition linked to several genetic, metabolic, and growth disorders in which there is dysregulated insulin secretion. In infants, an inappropriately persistent hypoglycemic and hypoketotic state can cause severe brain injury leading to epilepsy, cerebral palsy, and neurodevelopmental disabilities due to the lack of glucose and ketone substrate to serve as fuel for the developing brain. The most common cause of persistent hypoglycemia in neonates and children has been found to be congenital hyperinsulinism. Here, we report a child with a unique presentation, found to have a novel genetic variant as the underlying cause of hyperinsulinism. This case study highlights the importance of maintaining a broad differential and considering a diagnosis of congenital hyperinsulinism in a baby with poor feeding in the newborn period. Recognizing and treating congenital hyperinsulinism is essential to prevent potential neurological sequelae from recurrent, severe hypoglycemia.

Mosaic de novo SNRPN gene variant associated with Prader-Willi syndrome.

BACKGROUND: Prader-Willi syndrome (PWS) is an imprinting disorder caused by the absence of paternal expressed genes in the Prader-Willi critical region (PWCR) on chromosome 15q11.2-q13. Three molecular mechanisms have been known to cause PWS, including a deletion in the PWCR, uniparental disomy 15 and imprinting defects. RESULTS: We report the first case of PWS associated with a single-nucleotide SNRPN variant in a 10-year-old girl presenting with clinical features consistent with PWS, including infantile hypotonia and feeding difficulty, developmental delay with cognitive impairment, excessive eating with central obesity, sleep disturbances, skin picking and related behaviour issues. Whole-exome sequencing revealed a de novo mosaic nonsense variant of the SNRPN gene (c.73C>T, p.R25X) in 10% of DNA isolated from buccal cells and 19% of DNA from patient-derived lymphoblast cells. DNA methylation study did not detect an abnormal methylation pattern in the SNRPN locus. Parental origin studies showed a paternal source of an intronic single-nucleotide polymorphism within the locus in proximity to the SNRPN variant. CONCLUSIONS: This is the first report that provides evidence of a de novo point mutation of paternal origin in SNRPN as a new disease-causing mechanism for PWS. This finding suggests that gene sequencing should be considered as part of the diagnostic workup in patients with clinical suspicion of PWS.

A KCNB1 gain of function variant causes developmental delay and speech apraxia but not seizures.

Objective: Numerous pathogenic variants in KCNB1, which encodes the voltage-gated potassium channel, KV2.1, are linked to developmental and epileptic encephalopathies and associated with loss-of-function, -regulation, and -expression of the channel. Here we describe a novel de novo variant (P17T) occurring in the KV2.1 channel that is associated with a gain-of-function (GoF), with altered steady-state inactivation and reduced sensitivity to the selective toxin, guanxitoxin-1E and is clinically associated with neurodevelopmental disorders, without seizures. Methods: The autosomal dominant variant was identified using whole exome sequencing (WES). The functional effects of the KCNB1 variant on the encoded KV2.1 channel were investigated using whole-cell patch-clamp recordings. Results: We identified a de novo missense variant in the coding region of the KCNB1 gene, c.49C>A which encodes a p.P17T mutation in the N-terminus of the voltage-gated, KV2.1 potassium channel. Electrophysiological studies measuring the impact of the variant on the functional properties of the channel, identified a gain of current, rightward shifts in the steady-state inactivation curve and reduced sensitivity to the blocker, guanxitoxin-1E. Interpretation: The clinical evaluation of this KCNB1 mutation describes a novel variant that is associated with global developmental delays, mild hypotonia and joint laxity, but without seizures. Most of the phenotypic features described are reported for other variants of the KCNB1 gene. However, the absence of early-onset epileptic disorders is a much less common occurrence. This lack of seizure activity may be because other variants reported have resulted in loss-of-function of the encoded KV2.1 potassium channel, whereas this variant causes a gain-of-function.

Novel variants in KAT6B spectrum of disorders expand our knowledge of clinical manifestations and molecular mechanisms.

The phenotypic variability associated with pathogenic variants in Lysine Acetyltransferase 6B (KAT6B, a.k.a. MORF, MYST4) results in several interrelated syndromes including Say-Barber-Biesecker-Young-Simpson Syndrome and Genitopatellar Syndrome. Here we present 20 new cases representing 10 novel KAT6B variants. These patients exhibit a range of clinical phenotypes including intellectual disability, mobility and language difficulties, craniofacial dysmorphology, and skeletal anomalies. Given the range of features previously described for KAT6B-related syndromes, we have identified additional phenotypes including concern for keratoconus, sensitivity to light or noise, recurring infections, and fractures in greater numbers than previously reported. We surveyed clinicians to qualitatively assess the ways families engage with genetic counselors upon diagnosis. We found that 56% (10/18) of individuals receive diagnoses before the age of 2 years (median age = 1.96 years), making it challenging to address future complications with limited accessible information and vast phenotypic severity. We used CRISPR to introduce truncating variants into the KAT6B gene in model cell lines and performed chromatin accessibility and transcriptome sequencing to identify key dysregulated pathways. This study expands the clinical spectrum and addresses the challenges to management and genetic counseling for patients with KAT6B-related disorders.

Expanding the phenotype of ASXL3-related syndrome: A comprehensive description of 45 unpublished individuals with inherited and de novo pathogenic variants in ASXL3.

The study aimed at widening the clinical and genetic spectrum of ASXL3-related syndrome, a neurodevelopmental disorder, caused by truncating variants in the ASXL3 gene. In this international collaborative study, we have undertaken a detailed clinical and molecular analysis of 45 previously unpublished individuals with ASXL3-related syndrome, as well as a review of all previously published individuals. We have reviewed the rather limited functional characterization of pathogenic variants in ASXL3 and discuss current understanding of the consequences of the different ASXL3 variants. In this comprehensive analysis of ASXL3-related syndrome, we define its natural history and clinical evolution occurring with age. We report familial ASXL3 pathogenic variants, characterize the phenotype in mildly affected individuals and discuss nonpenetrance. We also discuss the role of missense variants in ASXL3. We delineate a variable but consistent phenotype. The most characteristic features are neurodevelopmental delay with consistently limited speech, significant neuro-behavioral issues, hypotonia, and feeding difficulties. Distinctive features include downslanting palpebral fissures, hypertelorism, tubular nose with a prominent nasal bridge, and low-hanging columella. The presented data will inform clinical management of individuals with ASXL3-related syndrome and improve interpretation of new ASXL3 sequence variants.

Nonlethal presentations of CYP26B1-related skeletal anomalies and multiple synostoses syndrome.

Retinoic acid exposures as well as defects in the retinoic acid-degrading enzyme CYP26B1 have teratogenic effects on both limb and craniofacial skeleton. An initial report of four individuals described a syndrome of fetal and infantile lethality with craniosynostosis and skeletal anomalies caused by homozygous pathogenic missense variants in CYP26B1. In contrast, a 22-year-old female was reported with a homozygous missense pathogenic variant in CYP26B1 with complex multisuture craniosynostosis and intellectual disability, suggesting that in some cases, biallelic pathogenic variants of CYP26B1 may be compatible with life. Here we describe four additional living individuals from two families with compound heterozygous pathogenic missense variants in CYP26B1. Structural assessment of these additional missense variants places them further from the catalytic site and supports a model consistent with milder nonlethal disease. In addition to previously reported findings of multisuture craniosynostosis, conductive hearing loss, joint contractures, long slender fingers, camptodactly, broad fingertips, and developmental delay/intellectual disability, skeletal imaging in our cases also revealed gracile long bones, gracile ribs, radioulnar synostosis, and carpal and/or tarsal fusions. These individuals broaden the phenotypic range of biallelic pathogenic variants in CYPB26B1 and most significantly clarify that mortality can range from perinatal lethality to survival into adulthood.

Pathogenic variants in SMARCA5, a chromatin remodeler, cause a range of syndromic neurodevelopmental features.

Intellectual disability encompasses a wide spectrum of neurodevelopmental disorders, with many linked genetic loci. However, the underlying molecular mechanism for more than 50% of the patients remains elusive. We describe pathogenic variants in SMARCA5, encoding the ATPase motor of the ISWI chromatin remodeler, as a cause of a previously unidentified neurodevelopmental disorder, identifying 12 individuals with de novo or dominantly segregating rare heterozygous variants. Accompanying phenotypes include mild developmental delay, frequent postnatal short stature and microcephaly, and recurrent dysmorphic features. Loss of function of the SMARCA5 Drosophila ortholog Iswi led to smaller body size, reduced sensory dendrite complexity, and tiling defects in larvae. In adult flies, Iswi neural knockdown caused decreased brain size, aberrant mushroom body morphology, and abnormal locomotor function. Iswi loss of function was rescued by wild-type but not mutant SMARCA5. Our results demonstrate that SMARCA5 pathogenic variants cause a neurodevelopmental syndrome with mild facial dysmorphia.

Pediatric Cushing syndrome: An early sign of an underling cancer predisposition syndrome.

Beckwith-Wiedemann syndrome (BWS) is a genetic overgrowth and cancer predisposition syndrome that can be associated with a spectrum of clinical features including isolated lateralized overgrowth, macrosomia, macroglossia, organomegaly, omphalocele/umbilical hernia, and distinct facial features. Because of a range of clinical presentations and molecular defects involving Chromosome 11p15, many cases will fall within what is now being defined as the Beckwith-Wiedemann spectrum (BWSp). Cushing syndrome (CS) in infants is a rare neuroendocrinological disease associated with hypercortisolism that has rarely been reported in patients with BWS. Here, we describe the first case of a 5-month-old male with CS secondary to paternal uniparental disomy of Chromosome 11p without additional clinical signs or symptoms of BWS. This case continues to expand the phenotypic spectrum of BWSp.

Whole genome sequencing identifies a cryptic SOX9 regulatory element duplication underlying a case of 46,XX ovotesticular difference of sexual development.

Ovotesticular differences of sexual development (OT-DSD) are rare genetic variances defined by the coexistence of both testicular and ovarian tissues. Various molecular etiologies including SRY translocation or SOX9 pathogenic variants with different modes of inheritance have been associated with 46,XX OT-DSD. Here we describe a child diagnosed with SRY-negative 46,XX OT-DSD after completing a series of complex clinical genetic analyses, including chromosomal microarray, DSD gene panel (sequencing and deletion/duplication analysis), whole exome sequencing, and whole genome sequencing. Of these, only whole genome sequencing reported a pathogenic duplication in a non-coding region that contains the RevSex regulatory element, which modifies SOX9 expression and is associated with 46,XX OT-DSD and complete sex reversal. This is the first clinical RevSex duplication detected by clinical whole genome sequencing. We highlight the utility of whole genome sequencing in shortening the diagnostic odyssey and the importance of optimal counseling through a team-based multi-specialty approach for patients with DSDs.

Thinking outside "The Box": Case-based didactics for medical education and the instructional legacy of Dr John M. Graham, Jr.

The increasing demand for advanced genomic services has finally come to the attention of healthcare systems and stakeholders who are now eager to find creative solutions to increase the pool of genomic literate providers. Training in genetics and dysmorphology has historically been conducted as a self-driven practice in pattern recognition, ideally within a formal or informal apprenticeship supervised by a master diagnostician. In recent times, case-based learning, framed by flipped classroom pedagogy have become the preferred teaching methods for complex medical topics such as genetics and genomics. To illuminate this perspective, our article was written in honor of the teaching style and pedagogy of Dr John M. Graham Jr and his lifelong commitment to medical education and mentoring.

Pathogenic variants in CDH11 impair cell adhesion and cause Teebi hypertelorism syndrome.

Teebi hypertelorism syndrome (THS; OMIM 145420) is a rare craniofacial disorder characterized by hypertelorism, prominent forehead, short nose with broad or depressed nasal root. Some cases of THS have been attributed to SPECC1L variants. Homozygous variants in CDH11 truncating the transmembrane and intracellular domains have been implicated in Elsahy-Waters syndrome (EWS; OMIM 211380) with hypertelorism. We report THS due to CDH11 heterozygous missense variants on 19 subjects from 9 families. All affected residues in the extracellular region of Cadherin-11 (CHD11) are highly conserved across vertebrate species and classical cadherins. Six of the variants that cluster around the EC2-EC3 and EC3-EC4 linker regions are predicted to affect Ca2+ binding that is required for cadherin stability. Two of the additional variants [c.164G > C, p.(Trp55Ser) and c.418G > A, p.(Glu140Lys)] are also notable as they are predicted to directly affect trans-homodimer formation. Immunohistochemical study demonstrates that CDH11 is strongly expressed in human facial mesenchyme. Using multiple functional assays, we show that five variants from the EC1, EC2-EC3 linker, and EC3 regions significantly reduced the cell-substrate trans adhesion activity and one variant from EC3-EC4 linker results in changes in cell morphology, focal adhesion, and migration, suggesting dominant negative effect. Characteristic features in this cohort included depressed nasal root, cardiac and umbilical defects. These features distinguished this phenotype from that seen in SPECC1L-related hypertelorism syndrome and CDH11-related EWS. Our results demonstrate heterozygous variants in CDH11, which decrease cell-cell adhesion and increase cell migratory behavior, cause a form of THS, as termed CDH11-related THS.

Further delineation of van den Ende-Gupta syndrome: Genetic heterogeneity and overlap with congenital heart defects and skeletal malformations syndrome.

Van den Ende-Gupta syndrome (VDEGS) is a rare autosomal recessive condition characterized by distinctive facial and skeletal features, and in most affected persons, by biallelic pathogenic variants in SCARF2. We review the type and frequency of the clinical features in 36 reported individuals with features of VDEGS, 15 (42%) of whom had known pathogenic variants in SCARF2, 6 (16%) with negative SCARF2 testing, and 15 (42%) not tested. We also report three new individuals with pathogenic variants in SCARF2 and clinical features of VDEGS. Of the six persons without known pathogenic variants in SCARF2, three remain unsolved despite extensive genetic testing. Three were found to have pathogenic ABL1 variants using whole exome sequencing (WES) or whole genome sequencing (WGS). Their phenotype was consistent with the congenital heart disease and skeletal malformations syndrome (CHDSKM), which has been associated with ABL1 variants. Of the three unsolved cases, two were brothers who underwent WGS and targeted long-range sequencing of both SCARF2 and ABL1, and the third person who underwent WES and RNA sequencing for SCARF2. Because these affected individuals with classical features of VDEGS lacked a detectable pathogenic SCARF2 variant, genetic heterogeneity is likely. Our study shows the importance of performing genetic testing on individuals with the VDEGS "phenotype," either as a targeted gene analysis (SCARF2, ABL1) or WES/WGS. Additionally, individuals with the combination of arachnodactyly and blepharophimosis should undergo echocardiography while awaiting results of molecular testing due to the overlapping physical features of VDEGS and CHDSKM.