The phenotype of MEGF8-related Carpenter syndrome (CRPT2) is refined through the identification of eight new patients.

Carpenter syndrome (CRPTS) is a rare autosomal recessive condition caused by biallelic variants in genes that encode negative regulators of hedgehog signalling (RAB23 [CRPT1] or, more rarely, MEGF8 [CRPT2]), and is characterised by craniosynostosis, polysyndactyly, and other congenital abnormalities. We describe a further six families comprising eight individuals with MEGF8-associated CRPT2, increasing the total number of reported cases to fifteen, and refine the phenotype of CRPT2 compared to CRPT1. The core features of craniosynostosis, polysyndactyly and (in males) cryptorchidism are almost universal in both CRPT1 and CRPT2. However, laterality defects are present in nearly half of those with MEGF8-associated CRPT2, but are rare in RAB23-associated CRPT1. Craniosynostosis in CRPT2 commonly involves a single midline suture in comparison to the multi-suture craniosynostosis characteristic of CRPT1. No patient to date has carried two MEGF8 gene alterations that are both predicted to lead to complete loss-of-function, suggesting that a variable degree of residual MEGF8 activity may be essential for viability and potentially contributing to variable phenotypic severity. These data refine the phenotypic spectrum of CRPT2 in comparison to CRPT1 and more than double the number of likely pathogenic MEGF8 variants in this rare disorder.

Personal journeys to and in human genetics and dysmorphology.

Genetics has become a critical component of medicine over the past five to six decades. Alongside genetics, a relatively new discipline, dysmorphology, has also begun to play an important role in providing critically important diagnoses to individuals and families. Both have become indispensable to unraveling rare diseases. Almost every medical specialty relies on individuals experienced in these specialties to provide diagnoses for patients who present themselves to other doctors. Additionally, both specialties have become reliant on molecular geneticists to identify genes associated with human disorders. Many of the medical geneticists, dysmorphologists, and molecular geneticists traveled a circuitous route before arriving at the position they occupied. The purpose of collecting the memoirs contained in this article was to convey to the reader that many of the individuals who contributed to the advancement of genetics and dysmorphology since the late 1960s/early 1970s traveled along a journey based on many chances taken, replying to the necessities they faced along the way before finding full enjoyment in the practice of medical and human genetics or dysmorphology. Additionally, and of equal importance, all exhibited an ability to evolve with their field of expertise as human genetics became human genomics with the development of novel technologies.

Ear anomalies and hearing loss in patients with VACTERL association and the effect of maternal diabetes.

VACTERL association is typically defined as the presence of three components among these birth defects: vertebral anomalies, anal atresia, cardiac anomalies, esophageal atresia/tracheoesophageal fistula (EA/TEF), renal anomalies, and limb defects. There is increasing recognition that VACTERL and other recurrent constellations of embryonic development often overlap clinically and might share pathogenesis. We conducted a comprehensive chart review of a large patient population with VACTERL association from two tertiary care centers in California. We included patients with incomplete VACTERL expression, which we denoted as "partial VACTERL" (pVACTERL). We assessed the occurrence of craniofacial (CF) findings in these two groups and the combined cohort. We collected data on potential risk factors and demographic information such as sex, Hispanic ancestry, pregnancy complications, and maternal age. The study included 409 participants, of whom 263 had VACTERL and 146 pVACTERL. CF abnormalities were found in 17.3% of VACTERL patients and 9.4% of pVACTERL patients. In the VACTERL group, ear anomalies were found in 10.2%, microtia in 5.9%, hearing loss (HL) in 13.90%, and orofacial clefts in 3.1%. In the pVACTERL group, ear anomalies were found in 7.2%, microtia in 5.0%, HL in 9.3%, and orofacial cleft in 2.2%. Maternal diabetes significantly increased the risk for HL in VACTERL (odds ratio [OR]: 3.71, 95% confidence interval [CI]: 1.5-7.3) and pVACTERL patients (OR: 6.7, 95% CI: 1.70-23.4). Poorly controlled maternal diabetes significantly increased the risk for all the outcomes in VACTERL patients including CF anomalies (OR: 4.2, 95% CI: 1.9-9.6), ear anomalies (OR: 4.7, 95% CI: 1.8-11.8), microtia (OR: 5.4, 95% CI: 1.7-16.6), and HL (OR: 8.1, 95% CI: 3.4-19.4). Twin status was significantly associated with the occurrence of microtia (p = 0.038) in VACTERL patients. Occurrence of CF features, particularly ear anomalies, microtia, and HL, might be considered as part of phenotypic diversity of VACTERL association. Diabetes and twinning might appear to play a role in increasing the risk for this phenotype in VACTERL association.

Jansen-de Vries syndrome: Expansion of the PPM1D clinical and phenotypic spectrum in 34 families.

Jansen-de Vries syndrome (JdVS) is a neurodevelopmental condition attributed to pathogenic variants in Exons 5 and 6 of PPM1D. As the full phenotypic spectrum and natural history remain to be defined, we describe a large cohort of children and adults with JdVS. This is a retrospective cohort study of 37 individuals from 34 families with disease-causing variants in PPM1D leading to JdVS. Clinical data were provided by treating physicians and/or families. Of the 37 individuals, 27 were male and 10 female, with median age 8.75 years (range 8 months to 62 years). Four families document autosomal dominant transmission, and 32/34 probands were diagnosed via exome sequencing. The facial gestalt, including a broad forehead and broad mouth with a thin and tented upper lip, was most recognizable between 18 and 48 months of age. Common manifestations included global developmental delay (35/36, 97%), hypotonia (25/34, 74%), short stature (14/33, 42%), constipation (22/31, 71%), and cyclic vomiting (6/35, 17%). Distinctive personality traits include a hypersocial affect (21/31, 68%) and moderate-to-severe anxiety (18/28, 64%). In conclusion, JdVS is a clinically recognizable neurodevelopmental syndrome with a characteristic personality and distinctive facial features. The association of pathogenic variants in PPM1D with cyclic vomiting bears not only medical attention but also further pathogenic and mechanistic evaluation.

Diagnostic yield of pediatric and prenatal exome sequencing in a diverse population.

The diagnostic yield of exome sequencing (ES) has primarily been evaluated in individuals of European ancestry, with less focus on underrepresented minority (URM) and underserved (US) patients. We evaluated the diagnostic yield of ES in a cohort of predominantly US and URM pediatric and prenatal patients suspected to have a genetic disorder. Eligible pediatric patients had multiple congenital anomalies and/or neurocognitive disabilities and prenatal patients had one or more structural anomalies, disorders of fetal growth, or fetal effusions. URM and US patients were prioritized for enrollment and underwent ES at a single academic center. We identified definitive positive or probable positive results in 201/845 (23.8%) patients, with a significantly higher diagnostic rate in pediatric (26.7%) compared to prenatal patients (19.0%) (P = 0.01). For both pediatric and prenatal patients, the diagnostic yield and frequency of inconclusive findings did not differ significantly between URM and non-URM patients or between patients with US status and those without US status. Our results demonstrate a similar diagnostic yield of ES between prenatal and pediatric URM/US patients and non-URM/US patients for positive and inconclusive results. These data support the use of ES to identify clinically relevant variants in patients from diverse populations.

Deleterious, protein-altering variants in the transcriptional coregulator ZMYM3 in 27 individuals with a neurodevelopmental delay phenotype.

Neurodevelopmental disorders (NDDs) result from highly penetrant variation in hundreds of different genes, some of which have not yet been identified. Using the MatchMaker Exchange, we assembled a cohort of 27 individuals with rare, protein-altering variation in the transcriptional coregulator ZMYM3, located on the X chromosome. Most (n = 24) individuals were males, 17 of which have a maternally inherited variant; six individuals (4 male, 2 female) harbor de novo variants. Overlapping features included developmental delay, intellectual disability, behavioral abnormalities, and a specific facial gestalt in a subset of males. Variants in almost all individuals (n = 26) are missense, including six that recurrently affect two residues. Four unrelated probands were identified with inherited variation affecting Arg441, a site at which variation has been previously seen in NDD-affected siblings, and two individuals have de novo variation resulting in p.Arg1294Cys (c.3880C>T). All variants affect evolutionarily conserved sites, and most are predicted to damage protein structure or function. ZMYM3 is relatively intolerant to variation in the general population, is widely expressed across human tissues, and encodes a component of the KDM1A-RCOR1 chromatin-modifying complex. ChIP-seq experiments on one variant, p.Arg1274Trp, indicate dramatically reduced genomic occupancy, supporting a hypomorphic effect. While we are unable to perform statistical evaluations to definitively support a causative role for variation in ZMYM3, the totality of the evidence, including 27 affected individuals, recurrent variation at two codons, overlapping phenotypic features, protein-modeling data, evolutionary constraint, and experimentally confirmed functional effects strongly support ZMYM3 as an NDD-associated gene.

Dominant ARF3 variants disrupt Golgi integrity and cause a neurodevelopmental disorder recapitulated in zebrafish.

Vesicle biogenesis, trafficking and signaling via Endoplasmic reticulum-Golgi network support essential developmental processes and their disruption lead to neurodevelopmental disorders and neurodegeneration. We report that de novo missense variants in ARF3, encoding a small GTPase regulating Golgi dynamics, cause a developmental disease in humans impairing nervous system and skeletal formation. Microcephaly-associated ARF3 variants affect residues within the guanine nucleotide binding pocket and variably perturb protein stability and GTP/GDP binding. Functional analysis demonstrates variably disruptive consequences of ARF3 variants on Golgi morphology, vesicles assembly and trafficking. Disease modeling in zebrafish validates further the dominant behavior of the mutants and their differential impact on brain and body plan formation, recapitulating the variable disease expression. In-depth in vivo analyses traces back impaired neural precursors' proliferation and planar cell polarity-dependent cell movements as the earliest detectable effects. Our findings document a key role of ARF3 in Golgi function and demonstrate its pleiotropic impact on development.

Exome sequencing identifies genetic variants in anophthalmia and microphthalmia.

Anophthalmia and microphthalmia (A/M) are rare birth defects affecting up to 2 per 10,000 live births. These conditions are manifested by the absence of an eye or reduced eye volumes within the orbit leading to vision loss. Although clinical case series suggest a strong genetic component in A/M, few systematic investigations have been conducted on potential genetic contributions owing to low population prevalence. To overcome this challenge, we utilized DNA samples and data collected as part of the National Birth Defects Prevention Study (NBDPS). The NBDPS employed multi-center ascertainment of infants affected by A/M. We performed exome sequencing on 67 family trios and identified numerous genes affected by rare deleterious nonsense and missense variants in this cohort, including de novo variants. We identified 9 nonsense changes and 86 missense variants that are absent from the reference human population (Genome Aggregation Database), and we suggest that these are high priority candidate genes for A/M. We also performed literature curation, single cell transcriptome comparisons, and molecular pathway analysis on the candidate genes and performed protein structure modeling to determine the potential pathogenic variant consequences on PAX6 in this disease.

Clinical findings and a DNA methylation signature in kindreds with alterations in ZNF711.

ZNF711 is one of eleven zinc-finger genes on the X chromosome that have been associated with X-linked intellectual disability. This association is confirmed by the clinical findings in 20 new cases in addition to 11 cases previously reported. No consistent growth aberrations, craniofacial dysmorphology, malformations or neurologic findings are associated with alterations in ZNF711. The intellectual disability is typically mild and coexisting autism occurs in half of the cases. Carrier females show no manifestations. A ZNF711-specific methylation signature has been identified which can assist in identifying new cases and in confirming the pathogenicity of variants in the gene.

SUFU haploinsufficiency causes a recognisable neurodevelopmental phenotype at the mild end of the Joubert syndrome spectrum.

BACKGROUND: Joubert syndrome (JS) is a recessively inherited ciliopathy characterised by congenital ocular motor apraxia (COMA), developmental delay (DD), intellectual disability, ataxia, multiorgan involvement, and a unique cerebellar and brainstem malformation. Over 40 JS-associated genes are known with a diagnostic yield of 60%-75%.In 2018, we reported homozygous hypomorphic missense variants of the SUFU gene in two families with mild JS. Recently, heterozygous truncating SUFU variants were identified in families with dominantly inherited COMA, occasionally associated with mild DD and subtle cerebellar anomalies. METHODS: We reanalysed next generation sequencing (NGS) data in two cohorts comprising 1097 probands referred for genetic testing of JS genes. RESULTS: Heterozygous truncating and splice-site SUFU variants were detected in 22 patients from 17 families (1.5%) with strong male prevalence (86%), and in 8 asymptomatic parents. Patients presented with COMA, hypotonia, ataxia and mild DD, and only a third manifested intellectual disability of variable severity. Brain MRI showed consistent findings characterised by vermis hypoplasia, superior cerebellar dysplasia and subtle-to-mild abnormalities of the superior cerebellar peduncles. The same pattern was observed in two out of three tested asymptomatic parents. CONCLUSION: Heterozygous truncating or splice-site SUFU variants cause a novel neurodevelopmental syndrome encompassing COMA and mild JS, which likely represent overlapping entities. Variants can arise de novo or be inherited from a healthy parent, representing the first cause of JS with dominant inheritance and reduced penetrance. Awareness of this condition will increase the diagnostic yield of JS genetic testing, and allow appropriate counselling about prognosis, medical monitoring and recurrence risk.

The clinical and molecular spectrum of QRICH1 associated neurodevelopmental disorder.

De novo variants in QRICH1 (Glutamine-rich protein 1) has recently been reported in 11 individuals with intellectual disability (ID). The function of QRICH1 is largely unknown but it is likely to play a key role in the unfolded response of endoplasmic reticulum stress through transcriptional control of proteostasis. In this study, we present 27 additional individuals and delineate the clinical and molecular spectrum of the individuals (n = 38) with QRICH1 variants. The main clinical features were mild to moderate developmental delay/ID (71%), nonspecific facial dysmorphism (92%) and hypotonia (39%). Additional findings included poor weight gain (29%), short stature (29%), autism spectrum disorder (29%), seizures (24%) and scoliosis (18%). Minor structural brain abnormalities were reported in 52% of the individuals with brain imaging. Truncating or splice variants were found in 28 individuals and 10 had missense variants. Four variants were inherited from mildly affected parents. This study confirms that heterozygous QRICH1 variants cause a neurodevelopmental disorder including short stature and expands the phenotypic spectrum to include poor weight gain, scoliosis, hypotonia, minor structural brain anomalies, and seizures. Inherited variants from mildly affected parents are reported for the first time, suggesting variable expressivity.

ABHD16A deficiency causes a complicated form of hereditary spastic paraplegia associated with intellectual disability and cerebral anomalies.

ABHD16A (abhydrolase domain-containing protein 16A, phospholipase) encodes the major phosphatidylserine (PS) lipase in the brain. PS lipase synthesizes lysophosphatidylserine, an important signaling lipid that functions in the mammalian central nervous system. ABHD16A has not yet been associated with a human disease. In this report, we present a cohort of 11 affected individuals from six unrelated families with a complicated form of hereditary spastic paraplegia (HSP) who carry bi-allelic deleterious variants in ABHD16A. Affected individuals present with a similar phenotype consisting of global developmental delay/intellectual disability, progressive spasticity affecting the upper and lower limbs, and corpus callosum and white matter anomalies. Immunoblot analysis on extracts from fibroblasts from four affected individuals demonstrated little to no ABHD16A protein levels compared to controls. Our findings add ABHD16A to the growing list of lipid genes in which dysregulation can cause complicated forms of HSP and begin to describe the molecular etiology of this condition.

ATP1A2- and ATP1A3-associated early profound epileptic encephalopathy and polymicrogyria.

Constitutional heterozygous mutations of ATP1A2 and ATP1A3, encoding for two distinct isoforms of the Na+/K+-ATPase (NKA) alpha-subunit, have been associated with familial hemiplegic migraine (ATP1A2), alternating hemiplegia of childhood (ATP1A2/A3), rapid-onset dystonia-parkinsonism, cerebellar ataxia-areflexia-progressive optic atrophy, and relapsing encephalopathy with cerebellar ataxia (all ATP1A3). A few reports have described single individuals with heterozygous mutations of ATP1A2/A3 associated with severe childhood epilepsies. Early lethal hydrops fetalis, arthrogryposis, microcephaly, and polymicrogyria have been associated with homozygous truncating mutations in ATP1A2. We investigated the genetic causes of developmental and epileptic encephalopathies variably associated with malformations of cortical development in a large cohort and identified 22 patients with de novo or inherited heterozygous ATP1A2/A3 mutations. We characterized clinical, neuroimaging and neuropathological findings, performed in silico and in vitro assays of the mutations' effects on the NKA-pump function, and studied genotype-phenotype correlations. Twenty-two patients harboured 19 distinct heterozygous mutations of ATP1A2 (six patients, five mutations) and ATP1A3 (16 patients, 14 mutations, including a mosaic individual). Polymicrogyria occurred in 10 (45%) patients, showing a mainly bilateral perisylvian pattern. Most patients manifested early, often neonatal, onset seizures with a multifocal or migrating pattern. A distinctive, 'profound' phenotype, featuring polymicrogyria or progressive brain atrophy and epilepsy, resulted in early lethality in seven patients (32%). In silico evaluation predicted all mutations to be detrimental. We tested 14 mutations in transfected COS-1 cells and demonstrated impaired NKA-pump activity, consistent with severe loss of function. Genotype-phenotype analysis suggested a link between the most severe phenotypes and lack of COS-1 cell survival, and also revealed a wide continuum of severity distributed across mutations that variably impair NKA-pump activity. We performed neuropathological analysis of the whole brain in two individuals with polymicrogyria respectively related to a heterozygous ATP1A3 mutation and a homozygous ATP1A2 mutation and found close similarities with findings suggesting a mainly neural pathogenesis, compounded by vascular and leptomeningeal abnormalities. Combining our report with other studies, we estimate that ∼5% of mutations in ATP1A2 and 12% in ATP1A3 can be associated with the severe and novel phenotypes that we describe here. Notably, a few of these mutations were associated with more than one phenotype. These findings assign novel, 'profound' and early lethal phenotypes of developmental and epileptic encephalopathies and polymicrogyria to the phenotypic spectrum associated with heterozygous ATP1A2/A3 mutations and indicate that severely impaired NKA pump function can disrupt brain morphogenesis.

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.

A dyadic approach to the delineation of diagnostic entities in clinical genomics.

The delineation of disease entities is complex, yet recent advances in the molecular characterization of diseases provide opportunities to designate diseases in a biologically valid manner. Here, we have formalized an approach to the delineation of Mendelian genetic disorders that encompasses two distinct but inter-related concepts: (1) the gene that is mutated and (2) the phenotypic descriptor, preferably a recognizably distinct phenotype. We assert that only by a combinatorial or dyadic approach taking both of these attributes into account can a unitary, distinct genetic disorder be designated. We propose that all Mendelian disorders should be designated as "GENE-related phenotype descriptor" (e.g., "CFTR-related cystic fibrosis"). This approach to delineating and naming disorders reconciles the complexity of gene-to-phenotype relationships in a simple and clear manner yet communicates the complexity and nuance of these relationships.

Alternative genomic diagnoses for individuals with a clinical diagnosis of Dubowitz syndrome.

Dubowitz syndrome (DubS) is considered a recognizable syndrome characterized by a distinctive facial appearance and deficits in growth and development. There have been over 200 individuals reported with Dubowitz or a "Dubowitz-like" condition, although no single gene has been implicated as responsible for its cause. We have performed exome (ES) or genome sequencing (GS) for 31 individuals clinically diagnosed with DubS. After genome-wide sequencing, rare variant filtering and computational and Mendelian genomic analyses, a presumptive molecular diagnosis was made in 13/27 (48%) families. The molecular diagnoses included biallelic variants in SKIV2L, SLC35C1, BRCA1, NSUN2; de novo variants in ARID1B, ARID1A, CREBBP, POGZ, TAF1, HDAC8, and copy-number variation at1p36.11(ARID1A), 8q22.2(VPS13B), Xp22, and Xq13(HDAC8). Variants of unknown significance in known disease genes, and also in genes of uncertain significance, were observed in 7/27 (26%) additional families. Only one gene, HDAC8, could explain the phenotype in more than one family (N = 2). All but two of the genomic diagnoses were for genes discovered, or for conditions recognized, since the introduction of next-generation sequencing. Overall, the DubS-like clinical phenotype is associated with extensive locus heterogeneity and the molecular diagnoses made are for emerging clinical conditions sharing characteristic features that overlap the DubS phenotype.

The spectrum of brain malformations and disruptions in twins.

Twins have an increased risk for congenital malformations and disruptions, including defects in brain morphogenesis. We analyzed data on brain imaging, zygosity, sex, and fetal demise in 56 proband twins and 7 less affected co-twins with abnormal brain imaging and compared them to population-based data and to a literature series. We separated our series into malformations of cortical development (MCD, N = 39), cerebellar malformations without MCD (N = 13), and brain disruptions (N = 11). The MCD group included 37/39 (95%) with polymicrogyria (PMG), 8/39 (21%) with pia-ependymal clefts (schizencephaly), and 15/39 (38%) with periventricular nodular heterotopia (PNH) including 2 with PNH but not PMG. Cerebellar malformations were found in 19 individuals including 13 with a cerebellar malformation only and another 6 with cerebellar malformation and MCD. The pattern varied from diffuse cerebellar hypoplasia to classic Dandy-Walker malformation. Brain disruptions were seen in 11 individuals with hydranencephaly, porencephaly, or white matter loss without cysts. Our series included an expected statistically significant excess of monozygotic (MZ) twin pairs (22/41 MZ, 54%) compared to population data (482/1448 MZ, 33.3%; p = .0110), and an unexpected statistically significant excess of dizygotic (DZ) twins (19/41, 46%) compared to the literature cohort (1/46 DZ, 2%; p < .0001. Recurrent association with twin-twin transfusion syndrome, intrauterine growth retardation, and other prenatal factors support disruption of vascular perfusion as the most likely unifying cause.