A drosophila genetic resource of mutants to study mechanisms underlying human genetic diseases.

Invertebrate model systems are powerful tools for studying human disease owing to their genetic tractability and ease of screening. We conducted a mosaic genetic screen of lethal mutations on the Drosophila X chromosome to identify genes required for the development, function, and maintenance of the nervous system. We identified 165 genes, most of whose function has not been studied in vivo. In parallel, we investigated rare variant alleles in 1,929 human exomes from families with unsolved Mendelian disease. Genes that are essential in flies and have multiple human homologs were found to be likely to be associated with human diseases. Merging the human data sets with the fly genes allowed us to identify disease-associated mutations in six families and to provide insights into microcephaly associated with brain dysgenesis. This bidirectional synergism between fly genetics and human genomics facilitates the functional annotation of evolutionarily conserved genes involved in human health.

PLS3 missense variants affecting the actin-binding domains cause X-linked congenital diaphragmatic hernia and body-wall defects.

Congenital diaphragmatic hernia (CDH) is a relatively common and genetically heterogeneous structural birth defect associated with high mortality and morbidity. We describe eight unrelated families with an X-linked condition characterized by diaphragm defects, variable anterior body-wall anomalies, and/or facial dysmorphism. Using linkage analysis and exome or genome sequencing, we found that missense variants in plastin 3 (PLS3), a gene encoding an actin bundling protein, co-segregate with disease in all families. Loss-of-function variants in PLS3 have been previously associated with X-linked osteoporosis (MIM: 300910), so we used in silico protein modeling and a mouse model to address these seemingly disparate clinical phenotypes. The missense variants in individuals with CDH are located within the actin-binding domains of the protein but are not predicted to affect protein structure, whereas the variants in individuals with osteoporosis are predicted to result in loss of function. A mouse knockin model of a variant identified in one of the CDH-affected families, c.1497G>C (p.Trp499Cys), shows partial perinatal lethality and recapitulates the key findings of the human phenotype, including diaphragm and abdominal-wall defects. Both the mouse model and one adult human male with a CDH-associated PLS3 variant were observed to have increased rather than decreased bone mineral density. Together, these clinical and functional data in humans and mice reveal that specific missense variants affecting the actin-binding domains of PLS3 might have a gain-of-function effect and cause a Mendelian congenital disorder.

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.

Genotype-phenotype correlations in RHOBTB2-associated neurodevelopmental disorders.

PURPOSE: Missense variants clustering in the BTB domain region of RHOBTB2 cause a developmental and epileptic encephalopathy with early-onset seizures and severe intellectual disability. METHODS: By international collaboration, we assembled individuals with pathogenic RHOBTB2 variants and a variable spectrum of neurodevelopmental disorders. By western blotting, we investigated the consequences of missense variants in vitro. RESULTS: In accordance with previous observations, de novo heterozygous missense variants in the BTB domain region led to a severe developmental and epileptic encephalopathy in 16 individuals. Now, we also identified de novo missense variants in the GTPase domain in 6 individuals with apparently more variable neurodevelopmental phenotypes with or without epilepsy. In contrast to variants in the BTB domain region, variants in the GTPase domain do not impair proteasomal degradation of RHOBTB2 in vitro, indicating different functional consequences. Furthermore, we observed biallelic splice-site and truncating variants in 9 families with variable neurodevelopmental phenotypes, indicating that complete loss of RHOBTB2 is pathogenic as well. CONCLUSION: By identifying genotype-phenotype correlations regarding location and consequences of de novo missense variants in RHOBTB2 and by identifying biallelic truncating variants, we further delineate and expand the molecular and clinical spectrum of RHOBTB2-related phenotypes, including both autosomal dominant and recessive neurodevelopmental disorders.

Delayed diagnosis and racial bias in children with genetic conditions.

As more therapeutics for genetic conditions become available, the need for timely and equitable genetic diagnosis has become urgent. Using clinical cases, we consider the health system-, provider-, and patient-level factors that contribute to the delayed diagnosis of genetic conditions in pediatric patients from minority populations, leading to health disparities between racial groups. We then provide suggestions to address these factors, with the aim of improving minority health and access to genetic care for all children.

CHEDDA syndrome is an underrecognized neurodevelopmental disorder with a highly restricted ATN1 mutation spectrum.

We describe the clinical features of nine unrelated individuals with rare de novo missense or in-frame deletions/duplications within the "HX motif" of exon 7 of ATN1. We previously proposed that individuals with such variants should be considered as being affected by the syndromic condition of congenital hypotonia, epilepsy, developmental delay, and digital anomalies (CHEDDA), distinct from dentatorubral-pallidoluysian atrophy (DRPLA) secondary to expansion variants in exon 5 of ATN1. We confirm that the universal phenotypic features of CHEDDA are distinctive facial features and global developmental delay. Infantile hypotonia and minor hand and feet differences are common and can present as arthrogryposis. Common comorbidities include severe feeding difficulties, often requiring gastrostomy support, as well as visual and hearing impairments. Epilepsy and congenital malformations of the brain, heart, and genitourinary systems are frequent but not universal. Our study confirms the clinical entity of CHEDDA secondary to a mutational signature restricted to exon 7 of ATN1. We propose a clinical schedule for assessment upon diagnosis, surveillance, and early intervention including the potential of neuroimaging for prognostication.

Cornelia de Lange syndrome in diverse populations.

Cornelia de Lange syndrome (CdLS) is a dominant multisystemic malformation syndrome due to mutations in five genes-NIPBL, SMC1A, HDAC8, SMC3, and RAD21. The characteristic facial dysmorphisms include microcephaly, arched eyebrows, synophrys, short nose with depressed bridge and anteverted nares, long philtrum, thin lips, micrognathia, and hypertrichosis. Most affected individuals have intellectual disability, growth deficiency, and upper limb anomalies. This study looked at individuals from diverse populations with both clinical and molecularly confirmed diagnoses of CdLS by facial analysis technology. Clinical data and images from 246 individuals with CdLS were obtained from 15 countries. This cohort included 49% female patients and ages ranged from infancy to 37 years. Individuals were grouped into ancestry categories of African descent, Asian, Latin American, Middle Eastern, and Caucasian. Across these populations, 14 features showed a statistically significant difference. The most common facial features found in all ancestry groups included synophrys, short nose with anteverted nares, and a long philtrum with thin vermillion of the upper lip. Using facial analysis technology we compared 246 individuals with CdLS to 246 gender/age matched controls and found that sensitivity was equal or greater than 95% for all groups. Specificity was equal or greater than 91%. In conclusion, we present consistent clinical findings from global populations with CdLS while demonstrating how facial analysis technology can be a tool to support accurate diagnoses in the clinical setting. This work, along with prior studies in this arena, will assist in earlier detection, recognition, and treatment of CdLS worldwide.

A recurrent mutation in MED12 leading to R961W causes Opitz-Kaveggia syndrome.

Opitz-Kaveggia syndrome (also known as FG syndrome) is an X-linked disorder characterized by mental retardation, relative macrocephaly, hypotonia and constipation. We report here that the original family for whom the condition is named and five other families have a recurrent mutation (2881C>T, leading to R961W) in MED12 (also called TRAP230 or HOPA), a gene located at Xq13 that functions as a thyroid receptor-associated protein in the Mediator complex.

Disruption of an EHMT1-associated chromatin-modification module causes intellectual disability.

Intellectual disability (ID) disorders are genetically and phenotypically highly heterogeneous and present a major challenge in clinical genetics and medicine. Although many genes involved in ID have been identified, the etiology is unknown in most affected individuals. Moreover, the function of most genes associated with ID remains poorly characterized. Evidence is accumulating that the control of gene transcription through epigenetic modification of chromatin structure in neurons has an important role in cognitive processes and in the etiology of ID. However, our understanding of the key molecular players and mechanisms in this process is highly fragmentary. Here, we identify a chromatin-modification module that underlies a recognizable form of ID, the Kleefstra syndrome phenotypic spectrum (KSS). In a cohort of KSS individuals without mutations in EHMT1 (the only gene known to be disrupted in KSS until now), we identified de novo mutations in four genes, MBD5, MLL3, SMARCB1, and NR1I3, all of which encode epigenetic regulators. Using Drosophila, we demonstrate that MBD5, MLL3, and NR1I3 cooperate with EHMT1, whereas SMARCB1 is known to directly interact with MLL3. We propose a highly conserved epigenetic network that underlies cognition in health and disease. This network should allow the design of strategies to treat the growing group of ID pathologies that are caused by epigenetic defects.

Biallelic variation in the choline and ethanolamine transporter FLVCR1 underlies a pleiotropic disease spectrum from adult neurodegeneration to severe developmental disorders.

FLVCR1 encodes Feline leukemia virus subgroup C receptor 1 (FLVCR1), a solute carrier (SLC) transporter within the Major Facilitator Superfamily. FLVCR1 is a widely expressed transmembrane protein with plasma membrane and mitochondrial isoforms implicated in heme, choline, and ethanolamine transport. While Flvcr1 knockout mice die in utero with skeletal malformations and defective erythropoiesis reminiscent of Diamond-Blackfan anemia, rare biallelic pathogenic FLVCR1 variants are linked to childhood or adult-onset neurodegeneration of the retina, spinal cord, and peripheral nervous system. We ascertained from research and clinical exome sequencing 27 individuals from 20 unrelated families with biallelic ultra-rare missense and predicted loss-of-function (pLoF) FLVCR1 variant alleles. We characterize an expansive FLVCR1 phenotypic spectrum ranging from adult-onset retinitis pigmentosa to severe developmental disorders with microcephaly, reduced brain volume, epilepsy, spasticity, and premature death. The most severely affected individuals, including three individuals with homozygous pLoF variants, share traits with Flvcr1 knockout mice and Diamond-Blackfan anemia including macrocytic anemia and congenital skeletal malformations. Pathogenic FLVCR1 missense variants primarily lie within transmembrane domains and reduce choline and ethanolamine transport activity compared with wild-type FLVCR1 with minimal impact on FLVCR1 stability or subcellular localization. Several variants disrupt splicing in a mini-gene assay which may contribute to genotype-phenotype correlations. Taken together, these data support an allele-specific gene dosage model in which phenotypic severity reflects residual FLVCR1 activity. This study expands our understanding of Mendelian disorders of choline and ethanolamine transport and demonstrates the importance of choline and ethanolamine in neurodevelopment and neuronal homeostasis.

Four new patients with Gomez-Lopez-Hernandez syndrome and proposed diagnostic criteria.

Gomez-Lopez-Hernandez syndrome (GLHS) is a rare neurocutaneous disorder. We are aware of thirty previously reported cases. We present four additional patients with this condition. Previously reported patients have shown the hallmark triad of rhombocephalosynapsis, trigeminal anesthesia, and bilateral parietal or parieto-occipital alopecia. Rhombencephalosynapsis consists of agenesis of the cerebellar vermis, fusion of the cerebellar hemispheres, and the dentate nuclei. The gene or genes responsible for GLHS remain unknown. Alopecia is seen in all previously reported cases of GLHS. Additional craniofacial findings such as low-set and posteriorly rotated ears, midface retrusion, craniosynostosis, and brachyturricephaly are also very common in this syndrome. Trigeminal anesthesia, reported in the original three patients, is seen in just over half of reported patients. Most patients with GLHS have motor delays, intellectual disability, and hypotonia. Unusual stereotypic movements of the head are seen in many patients with GLHS. Neuroimaging of patients with GLHS shows rhombencephalosynapsis is universally present, with ventriculomegaly/hydrocephalus and cerebellar hypoplasia being common. We propose that rhombencephalosynapsis and scalp alopecia are necessary, but by themselves not sufficient, for a diagnosis of GLHS. Additional findings of trigeminal anesthesia or one of two major craniofacial findings (brachycephaly and/or turricephaly or midface retrusion) are sufficient to make a diagnosis of GLHS. Additional categories of probable and possible GLHS are proposed for patients whose examination may be compatible with a diagnosis of GLHS, but CNS imaging has not yet been obtained. © 2013 Wiley Periodicals, Inc.

Mutations in LTBP4 cause a syndrome of impaired pulmonary, gastrointestinal, genitourinary, musculoskeletal, and dermal development.

We report recessive mutations in the gene for the latent transforming growth factor-beta binding protein 4 (LTBP4) in four unrelated patients with a human syndrome disrupting pulmonary, gastrointestinal, urinary, musculoskeletal, craniofacial, and dermal development. All patients had severe respiratory distress, with cystic and atelectatic changes in the lungs complicated by tracheomalacia and diaphragmatic hernia. Three of the four patients died of respiratory failure. Cardiovascular lesions were mild, limited to pulmonary artery stenosis and patent foramen ovale. Gastrointestinal malformations included diverticulosis, enlargement, tortuosity, and stenosis at various levels of the intestinal tract. The urinary tract was affected by diverticulosis and hydronephrosis. Joint laxity and low muscle tone contributed to musculoskeletal problems compounded by postnatal growth delay. Craniofacial features included microretrognathia, flat midface, receding forehead, and wide fontanelles. All patients had cutis laxa. Four of the five identified LTBP4 mutations led to premature termination of translation and destabilization of the LTBP4 mRNA. Impaired synthesis and lack of deposition of LTBP4 into the extracellular matrix (ECM) caused increased transforming growth factor-beta (TGF-beta) activity in cultured fibroblasts and defective elastic fiber assembly in all tissues affected by the disease. These molecular defects were associated with blocked alveolarization and airway collapse in the lung. Our results show that coupling of TGF-beta signaling and ECM assembly is essential for proper development and is achieved in multiple human organ systems by multifunctional proteins such as LTBP4.

Microdeletion 9q22.3 syndrome includes metopic craniosynostosis, hydrocephalus, macrosomia, and developmental delay.

Basal cell nevus syndrome (BCNS), also known as Gorlin syndrome (OMIM #109400) is a well-described rare autosomal dominant condition due to haploinsufficiency of PTCH1. With the availability of comparative genomic hybridization arrays, increasing numbers of individuals with microdeletions involving this locus are being identified. We present 10 previously unreported individuals with 9q22.3 deletions that include PTCH1. While 7 of the 10 patients (7 females, 3 males) did not meet strict clinical criteria for BCNS at the time of molecular diagnosis, almost all of the patients were too young to exhibit many of the diagnostic features. A number of the patients exhibited metopic craniosynostosis, severe obstructive hydrocephalus, and macrosomia, which are not typically observed in BCNS. All individuals older than a few months of age also had developmental delays and/or intellectual disability. Only facial features typical of BCNS, except in those with prominent midforeheads secondary to metopic craniosynostosis, were shared among the 10 patients. The deletions in these individuals ranged from 352 kb to 20.5 Mb in size, the largest spanning 9q21.33 through 9q31.2. There was significant overlap of the deleted segments among most of the patients. The smallest common regions shared among the deletions were identified in order to localize putative candidate genes that are potentially responsible for each of the non-BCNS features. These were a 929 kb region for metopic craniosynostosis, a 1.08 Mb region for obstructive hydrocephalus, and a 1.84 Mb region for macrosomia. Additional studies are needed to further characterize the candidate genes within these regions.

Discovery of a novel non-steroidal GR antagonist with in vivo efficacy in the olanzapine-induced weight gain model in the rat.

We report the optimization of a series of non-steroidal GR antagonists that led to the identification of compound 7. This compound is efficacious when dosed orally in an olanzapine-induced weight gain model in rats.

ANKLE2-related microcephaly: A variable microcephaly syndrome resembling Zika infection.

OBJECTIVE: This study delineates the clinical and molecular spectrum of ANKLE2-related microcephaly (MIC), as well as highlights shared pathological mechanisms between ANKLE2 and the Zika virus. METHODS: We identified 12 individuals with MIC and variants in ANKLE2 with a broad range of features. Probands underwent thorough phenotypic evaluations, developmental assessments, and anthropometric measurements. Brain imaging studies were systematically reviewed for developmental abnormalities. We functionally interrogated a subset of identified ANKLE2 variants in Drosophila melanogaster. RESULTS: All individuals had MIC (z-score ≤ -3), including nine with congenital MIC. We identified a broad range of brain abnormalities including simplified cortical gyral pattern, full or partial callosal agenesis, increased extra-axial spaces, hypomyelination, cerebellar vermis hypoplasia, and enlarged cisterna magna. All probands had developmental delays in at least one domain, with speech and language delays being the most common. Six probands had skin findings characteristic of ANKLE2 including hyper- and hypopigmented macules. Only one individual had scalp rugae. Functional characterization in Drosophila recapitulated the human MIC phenotype. Of the four variants tested, p.Val229Gly, p.Arg236*, and p.Arg536Cys acted as partial-loss-of-function variants, whereas the c.1421-1G>C splicing variant demonstrated a strong loss-of-function effect. INTERPRETATION: Deleterious variants in the ANKLE2 gene cause a unique MIC syndrome characterized by congenital or postnatal MIC, a broad range of structural brain abnormalities, and skin pigmentary changes. Thorough functional characterization has identified shared pathogenic mechanisms between ANKLE2-related MIC and congenital Zika virus infection. This study further highlights the importance of a thorough diagnostic evaluation including molecular diagnostic testing in individuals with MIC.

Rare missense and synonymous variants in UBE1 are associated with X-linked infantile spinal muscular atrophy.

X-linked infantile spinal muscular atrophy (XL-SMA) is an X-linked disorder presenting with the clinical features hypotonia, areflexia, and multiple congenital contractures (arthrogryposis) associated with loss of anterior horn cells and infantile death. To identify the XL-SMA disease gene, we performed large-scale mutation analysis in genes located between markers DXS8080 and DXS7132 (Xp11.3-Xq11.1). This resulted in detection of three rare novel variants in exon 15 of UBE1 that segregate with disease: two missense mutations (c.1617 G-->T, p.Met539Ile; c.1639 A-->G, p.Ser547Gly) present each in one XL-SMA family, and one synonymous C-->T substitution (c.1731 C-->T, p.Asn577Asn) identified in another three unrelated families. Absence of the missense mutations was demonstrated for 3550 and absence of the synonymous mutation was shown in 7914 control X chromosomes; therefore, these results yielded statistical significant evidence for the association of the synonymous substitution and the two missense mutations with XL-SMA (p = 2.416 x 10(-10), p = 0.001815). We also demonstrated that the synonymous C-->T substitution leads to significant reduction of UBE1 expression and alters the methylation pattern of exon 15, implying a plausible role of this DNA element in developmental UBE1 expression in humans. Our observations indicate first that XL-SMA is part of a growing list of neurodegenerative disorders associated with defects in the ubiquitin-proteasome pathway and second that synonymous C-->T transitions might have the potential to affect gene expression.

Behavioral features in young adults with FG syndrome (Opitz-Kaveggia syndrome).

Opitz and Kaveggia [Opitz and Kaveggia (1974); Z Kinderheilkd 117:1-18] reported on a family of five affected males with distinctive facial appearance, mental retardation, macrocephaly, imperforate anus, and hypotonia. Risheg et al. [Risheg et al. (2007); Nature Genetics 39:451-453] identified an identical mutation (p.R961W) in MED12 in six families with Opitz-Kaveggia syndrome, including a surviving affected man from the original family reported in 1974. The previously described behavior phenotype of hyperactivity, affability, and excessive talkativeness is very frequent in young boys with FG syndrome, along with socially oriented, attention-seeking behaviors. We present case studies of five adult males who were previously published with the clinical diagnosis of FG syndrome and then subsequently proven by Risheg et al. [Risheg et al. (2007); Nature Genetics 39:451-453] to have the recurrent p.R961W mutation. These individuals had episodic and longstanding behavior patterns, sometimes aggressive or self-abusing, that occurred more frequently in puberty and early adulthood. We try to describe the triggers for these behaviors, indicate how these behaviors change with advancing age, and suggest specific recommendations and interventional strategies based on the clinical histories of affected adolescent males with FG syndrome [Graham et al., 2008; Clark et al., 2009]. Young men who exhibit these behaviors may benefit from a careful examination to detect medical problems, use of mood stabilizers if needed, and/or behavioral intervention. The transition to a community living situation can be challenging without careful planning and timely behavioral intervention. They remain impulsive and can have aggressive outbursts when making the transition to adult life, but these challenges can be managed, as demonstrated by these clinical histories.

Overlapping spectra of SMAD4 mutations in juvenile polyposis (JP) and JP-HHT syndrome.

Juvenile polyposis (JP) and hereditary hemorrhagic telangiectasia (HHT) are clinically distinct diseases caused by mutations in SMAD4 and BMPR1A (for JP) and endoglin and ALK1 (for HHT). Recently, a combined syndrome of JP-HHT was described that is also caused by mutations in SMAD4. Although both JP and JP-HHT are caused by SMAD4 mutations, a possible genotype:phenotype correlation was noted as all of the SMAD4 mutations in the JP-HHT patients were clustered in the COOH-terminal MH2 domain of the protein. If valid, this correlation would provide a molecular explanation for the phenotypic differences, as well as a pre-symptomatic diagnostic test to distinguish patients at risk for the overlapping but different clinical features of the disorders. In this study, we collected 19 new JP-HHT patients from which we identified 15 additional SMAD4 mutations. We also reviewed the literature for other reports of JP patients with HHT symptoms with confirmed SMAD4 mutations. Our combined results show that although the SMAD4 mutations in JP-HHT patients do show a tendency to cluster in the MH2 domain, mutations in other parts of the gene also cause the combined syndrome. Thus, any mutation in SMAD4 can cause JP-HHT. Any JP patient with a SMAD4 mutation is, therefore, at risk for the visceral manifestations of HHT and any HHT patient with SMAD4 mutation is at risk for early onset gastrointestinal cancer. In conclusion, a patient who tests positive for any SMAD4 mutation must be considered at risk for the combined syndrome of JP-HHT and monitored accordingly.