The pheromone darcin drives a circuit for innate and reinforced behaviours.

Organisms have evolved diverse behavioural strategies that enhance the likelihood of encountering and assessing mates1. Many species use pheromones to communicate information about the location, sexual and social status of potential partners2. In mice, the major urinary protein darcin-which is present in the urine of males-provides a component of a scent mark that elicits approach by females and drives learning3,4. Here we show that darcin elicits a complex and variable behavioural repertoire that consists of attraction, ultrasonic vocalization and urinary scent marking, and also serves as a reinforcer in learning paradigms. We identify a genetically determined circuit-extending from the accessory olfactory bulb to the posterior medial amygdala-that is necessary for all behavioural responses to darcin. Moreover, optical activation of darcin-responsive neurons in the medial amygdala induces both the innate and the conditioned behaviours elicited by the pheromone. These neurons define a topographically segregated population that expresses neuronal nitric oxide synthase. We suggest that this darcin-activated neural circuit integrates pheromonal information with internal state to elicit both variable innate behaviours and reinforced behaviours that may promote mate encounters and mate selection.

Unraveling female communication through scent marks in the Norway rat.

Chemical communication by females remains poorly understood, with most attention focused on female advertisement of sexual receptivity to males or mother-offspring communication. However, in social species, scents are likely to be important for mediating competition and cooperation between females determining individual reproductive success. Here, we explore chemical signaling by female laboratory rats (Rattus norvegicus) to test i) whether females target their deployment of scent information differentially according to their sexual receptivity and the genetic identity of both female and male conspecifics signaling in the local environment and ii) whether females are attracted to gain the same or different information from female scents compared to males. Consistent with targeting of scent information to colony members of similar genetic background, female rats increased scent marking in response to scents from females of the same strain. Females also suppressed scent marking in response to male scent from a genetically foreign strain while sexually receptive. Proteomic analysis of female scent deposits revealed a complex protein profile, contributed from several sources but dominated by clitoral gland secretion. In particular, female scent marks contained a series of clitoral-derived hydrolases and proteolytically truncated major urinary proteins (MUPs). Manipulated blends of clitoral secretion and urine from estrus females were strongly attractive to both sexes, while voided urine alone stimulated no interest. Our study reveals that information about female receptive status is shared between females as well as with males, while clitoral secretions containing a complex set of truncated MUPs and other proteins play a key role in female communication.

Germline homozygous missense DEPDC5 variants cause severe refractory early-onset epilepsy, macrocephaly and bilateral polymicrogyria.

DEPDC5 (DEP Domain-Containing Protein 5) encodes an inhibitory component of the mammalian target of rapamycin (mTOR) pathway and is commonly implicated in sporadic and familial focal epilepsies, both non-lesional and in association with focal cortical dysplasia. Germline pathogenic variants are typically heterozygous and inactivating. We describe a novel phenotype caused by germline biallelic missense variants in DEPDC5. Cases were identified clinically. Available records, including magnetic resonance imaging and electroencephalography, were reviewed. Genetic testing was performed by whole exome and whole-genome sequencing and cascade screening. In addition, immunohistochemistry was performed on skin biopsy. The phenotype was identified in nine children, eight of which are described in detail herein. Six of the children were of Irish Traveller, two of Tunisian and one of Lebanese origin. The Irish Traveller children shared the same DEPDC5 germline homozygous missense variant (p.Thr337Arg), whereas the Lebanese and Tunisian children shared a different germline homozygous variant (p.Arg806Cys). Consistent phenotypic features included extensive bilateral polymicrogyria, congenital macrocephaly and early-onset refractory epilepsy, in keeping with other mTOR-opathies. Eye and cardiac involvement and severe neutropenia were also observed in one or more patients. Five of the children died in infancy or childhood; the other four are currently aged between 5 months and 6 years. Skin biopsy immunohistochemistry was supportive of hyperactivation of the mTOR pathway. The clinical, histopathological and genetic evidence supports a causal role for the homozygous DEPDC5 variants, expanding our understanding of the biology of this gene.

Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals.

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope-labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [13C6]lysine or [2H2]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.

Rapid identification of mosquito species and age by mass spectrometric analysis.

BACKGROUND: A rapid, accurate method to identify and to age-grade mosquito populations would be a major advance in predicting the risk of pathogen transmission and evaluating the public health impact of vector control interventions. Whilst other spectrometric or transcriptomic methods show promise, current approaches rely on challenging morphological techniques or simple binary classifications that cannot identify the subset of the population old enough to be infectious. In this study, the ability of rapid evaporative ionisation mass spectrometry (REIMS) to identify the species and age of mosquitoes reared in the laboratory and derived from the wild was investigated. RESULTS: The accuracy of REIMS in identifying morphologically identical species of the Anopheles gambiae complex exceeded 97% using principal component/linear discriminant analysis (PC-LDA) and 84% based on random forest analysis. Age separation into 3 different age categories (1 day, 5-6 days, 14-15 days) was achieved with 99% (PC-LDA) and 91% (random forest) accuracy. When tested on wild mosquitoes from the UK, REIMS data could determine the species and age of the specimens with accuracies of 91 and 90% respectively. CONCLUSIONS: The accuracy of REIMS to resolve the species and age of Anopheles mosquitoes is comparable to that achieved by infrared spectroscopy approaches. The processing time and ease of use represent significant advantages over current, dissection-based methods. Importantly, the accuracy was maintained when using wild mosquitoes reared under differing environmental conditions, and when mosquitoes were stored frozen or desiccated. This high throughput approach thus has potential to conduct rapid, real-time monitoring of vector populations, providing entomological evidence of the impact of alternative interventions.

Fitness Costs of Female Competition Linked to Resource Defense and Relatedness of Competitors.

AbstractFemale reproductive success is often limited by access to resources, and this can lead to social competition both within and between kin groups. Theory predicts that both resource availability and relatedness should influence the fitness consequences of social competition. However, testing key predictions requires differentiating the effects of these two factors. Here, we achieve this experimentally by manipulating the social environment of house mice, a facultative communal breeding species with known kin discrimination ability. This allows us to investigate (1) the reproductive costs of defending a limited resource in response to cues of social competition and (2) whether such costs, or their potential mitigation via cooperative behavior, are influenced by the relatedness of competitors. Our results support the hypothesis that resource defense can be costly for females, potentially trading off against maternal investment. When the availability of protected nest sites was limited, subjects (1) were more active, (2) responded more strongly to simulated territory intrusions via competitive signaling, and (3) produced smaller weaned offspring. However, we found no evidence that the propensity for kin to cooperate was influenced by the relatedness of rivals. Communal breeding between sisters occurred independently of the relatedness of competitors and communally breeding sisters weaned fewer offspring when competing with unrelated females, despite our study being designed to prevent infanticide between kin groups. Our findings thus demonstrate that female competition has fitness costs and that associating with kin is beneficial to avoid negative fitness consequences of competing with nonkin, in addition to more widely recognized kin-selected benefits.

Revealing mechanisms of mating plug function under sexual selection.

Mating plugs are produced by many sexually reproducing animals and are hypothesized to promote male fertilization success under promiscuous mating. However, tests of this hypothesis have been constrained by an inability to discriminate ejaculates of different males in direct competition. Here, we use stable isotope labeling in vivo and proteomics to achieve this in a promiscuous rodent, Myodes glareolus We show that, although the first male's plug is usually dislodged, it can be retained throughout the second male's copulation. Retained plugs did not completely block rival sperm but did significantly limit their numbers. Differences in the number of each male's sperm progressing through the female reproductive tract were also explained by natural variation in the size of mating plugs and reproductive accessory glands from which major plug proteins originate. Relative sperm numbers in turn predicted the relative fertilization success of rival males. Our application of stable isotopes to label ejaculates resolves a longstanding debate by revealing how rodent mating plugs promote fertilization success under competitive conditions. This approach opens new opportunities to reveal cryptic mechanisms of postcopulatory sexual selection among diverse animal taxa.

Novel truncating mutations in CTNND1 cause a dominant craniofacial and cardiac syndrome.

CTNND1 encodes the p120-catenin (p120) protein, which has a wide range of functions, including the maintenance of cell-cell junctions, regulation of the epithelial-mesenchymal transition and transcriptional signalling. Due to advances in next-generation sequencing, CTNND1 has been implicated in human diseases including cleft palate and blepharocheilodontic (BCD) syndrome albeit only recently. In this study, we identify eight novel protein-truncating variants, six de novo, in 13 participants from nine families presenting with craniofacial dysmorphisms including cleft palate and hypodontia, as well as congenital cardiac anomalies, limb dysmorphologies and neurodevelopmental disorders. Using conditional deletions in mice as well as CRISPR/Cas9 approaches to target CTNND1 in Xenopus, we identified a subset of phenotypes that can be linked to p120-catenin in epithelial integrity and turnover, and additional phenotypes that suggest mesenchymal roles of CTNND1. We propose that CTNND1 variants have a wider developmental role than previously described and that variations in this gene underlie not only cleft palate and BCD but may be expanded to a broader velocardiofacial-like syndrome.

Clinical delineation, sex differences, and genotype-phenotype correlation in pathogenic KDM6A variants causing X-linked Kabuki syndrome type 2.

PURPOSE: The variant spectrum and the phenotype of X-linked Kabuki syndrome type 2 (KS2) are poorly understood. METHODS: Genetic and clinical details of new and published individuals with pathogenic KDM6A variants were compiled and analyzed. RESULTS: Sixty-one distinct pathogenic KDM6A variants (50 truncating, 11 missense) from 80 patients (34 males, 46 females) were identified. Missense variants clustered in the TRP 2, 3, 7 and Jmj-C domains. Truncating variants were significantly more likely to be de novo. Thirteen individuals had maternally inherited variants and one had a paternally inherited variant. Neonatal feeding difficulties, hypoglycemia, postnatal growth retardation, poor weight gain, motor delay, intellectual disability (ID), microcephaly, congenital heart anomalies, palate defects, renal malformations, strabismus, hearing loss, recurrent infections, hyperinsulinism, seizures, joint hypermobility, and gastroesophageal reflux were frequent clinical findings. Facial features of over a third of patients were not typical for KS. Males were significantly more likely to be born prematurely, have shorter stature, and severe developmental delay/ID. CONCLUSION: We expand the KDM6A variant spectrum and delineate the KS2 phenotype. We demonstrate that the variability of the KS2 phenotypic depends on sex and the variant type. We also highlight the overlaps and differences between the phenotypes of KS2 and KS1.

Syndromic Forms of Hyperinsulinaemic Hypoglycaemia-A 15-year follow-up Study.

OBJECTIVE: Hyperinsulinaemic hypoglycaemia (HH) is one of the commonest causes of hypoglycaemia in children. The molecular basis includes defects in pathways that regulate insulin release. Syndromic conditions like Beckwith-Wiedemann (BWS), Kabuki (KS) and Turner (TS) are known to be associated with a higher risk for HH. This systematic review of children with HH referred to a tertiary centre aims at estimating the frequency of a syndromic/multisystem condition to help address stratification of genetic analysis in infants with HH. METHODS: We performed a retrospective study of 69 patients with syndromic features and hypoglycaemia in a specialist centre from 2004 to 2018. RESULTS: Biochemical investigations confirmed HH in all the cases and several genetic diagnoses were established. Responsiveness to medications and the final outcome following medical treatment or surgery were studied. CONCLUSIONS: This study highlights the association of HH with a wide spectrum of syndromic diagnoses and that children with features suggestive of HH-associated syndromes should be monitored for hypoglycaemia. If hypoglycaemia is documented, they should also be screened for possible HH. Our data indicate that most syndromic forms of HH are diazoxide-responsive and that HH resolves over time; however, a significant percentage continues to require medications years after the onset of the disease. Early diagnosis of hyperinsulinism and initiation of treatment is important for preventing hypoglycaemic brain injury and intellectual disability.

Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease.

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in CHM in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease.

High Rate of Recurrent De Novo Mutations in Developmental and Epileptic Encephalopathies.

Developmental and epileptic encephalopathy (DEE) is a group of conditions characterized by the co-occurrence of epilepsy and intellectual disability (ID), typically with developmental plateauing or regression associated with frequent epileptiform activity. The cause of DEE remains unknown in the majority of cases. We performed whole-genome sequencing (WGS) in 197 individuals with unexplained DEE and pharmaco-resistant seizures and in their unaffected parents. We focused our attention on de novo mutations (DNMs) and identified candidate genes containing such variants. We sought to identify additional subjects with DNMs in these genes by performing targeted sequencing in another series of individuals with DEE and by mining various sequencing datasets. We also performed meta-analyses to document enrichment of DNMs in candidate genes by leveraging our WGS dataset with those of several DEE and ID series. By combining these strategies, we were able to provide a causal link between DEE and the following genes: NTRK2, GABRB2, CLTC, DHDDS, NUS1, RAB11A, GABBR2, and SNAP25. Overall, we established a molecular diagnosis in 63/197 (32%) individuals in our WGS series. The main cause of DEE in these individuals was de novo point mutations (53/63 solved cases), followed by inherited mutations (6/63 solved cases) and de novo CNVs (4/63 solved cases). De novo missense variants explained a larger proportion of individuals in our series than in other series that were primarily ascertained because of ID. Moreover, these DNMs were more frequently recurrent than those identified in ID series. These observations indicate that the genetic landscape of DEE might be different from that of ID without epilepsy.

Recurrent heterozygous PAX6 missense variants cause severe bilateral microphthalmia via predictable effects on DNA-protein interaction.

PURPOSE: Most classical aniridia is caused by PAX6 haploinsufficiency. PAX6 missense variants can be hypomorphic or mimic haploinsufficiency. We hypothesized that missense variants also cause previously undescribed disease by altering the affinity and/or specificity of PAX6 genomic interactions. METHODS: We screened PAX6 in 372 individuals with bilateral microphthalmia, anophthalmia, or coloboma (MAC) from the Medical Research Council Human Genetics Unit eye malformation cohort (HGUeye) and reviewed data from the Deciphering Developmental Disorders study. We performed cluster analysis on PAX6-associated ocular phenotypes by variant type and molecular modeling of the structural impact of 86 different PAX6 causative missense variants. RESULTS: Eight different PAX6 missense variants were identified in 17 individuals (15 families) with MAC, accounting for 4% (15/372) of our cohort. Seven altered the paired domain (p.[Arg26Gln]x1, p.[Gly36Val]x1, p.[Arg38Trp]x2, p.[Arg38Gln]x1, p.[Gly51Arg]x2, p.[Ser54Arg]x2, p.[Asn124Lys]x5) and one the homeodomain (p.[Asn260Tyr]x1). p.Ser54Arg and p.Asn124Lys were exclusively associated with severe bilateral microphthalmia. MAC-associated variants were predicted to alter but not ablate DNA interaction, consistent with the electrophoretic mobility shifts observed using mutant paired domains with well-characterized PAX6-binding sites. We found no strong evidence for novel PAX6-associated extraocular disease. CONCLUSION: Altering the affinity and specificity of PAX6-binding genome-wide provides a plausible mechanism for the worse-than-null effects of MAC-associated missense variants.

GATAD2B-associated neurodevelopmental disorder (GAND): clinical and molecular insights into a NuRD-related disorder.

PURPOSE: Determination of genotypic/phenotypic features of GATAD2B-associated neurodevelopmental disorder (GAND). METHODS: Fifty GAND subjects were evaluated to determine consistent genotypic/phenotypic features. Immunoprecipitation assays utilizing in vitro transcription-translation products were used to evaluate GATAD2B missense variants' ability to interact with binding partners within the nucleosome remodeling and deacetylase (NuRD) complex. RESULTS: Subjects had clinical findings that included macrocephaly, hypotonia, intellectual disability, neonatal feeding issues, polyhydramnios, apraxia of speech, epilepsy, and bicuspid aortic valves. Forty-one novelGATAD2B variants were identified with multiple variant types (nonsense, truncating frameshift, splice-site variants, deletions, and missense). Seven subjects were identified with missense variants that localized within two conserved region domains (CR1 or CR2) of the GATAD2B protein. Immunoprecipitation assays revealed several of these missense variants disrupted GATAD2B interactions with its NuRD complex binding partners. CONCLUSIONS: A consistent GAND phenotype was caused by a range of genetic variants in GATAD2B that include loss-of-function and missense subtypes. Missense variants were present in conserved region domains that disrupted assembly of NuRD complex proteins. GAND's clinical phenotype had substantial clinical overlap with other disorders associated with the NuRD complex that involve CHD3 and CHD4, with clinical features of hypotonia, intellectual disability, cardiac defects, childhood apraxia of speech, and macrocephaly.

Correction: GATAD2B-associated neurodevelopmental disorder (GAND): clinical and molecular insights into a NuRD-related disorder.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genotype-phenotype correlation at codon 1740 of SETD2.

The SET domain containing 2, histone lysine methyltransferase encoded by SETD2 is a dual-function methyltransferase for histones and microtubules and plays an important role for transcriptional regulation, genomic stability, and cytoskeletal functions. Specifically, SETD2 is associated with trimethylation of histone H3 at lysine 36 (H3K36me3) and methylation of α-tubulin at lysine 40. Heterozygous loss of function and missense variants have previously been described with Luscan-Lumish syndrome (LLS), which is characterized by overgrowth, neurodevelopmental features, and absence of overt congenital anomalies. We have identified 15 individuals with de novo variants in codon 1740 of SETD2 whose features differ from those with LLS. Group 1 consists of 12 individuals with heterozygous variant c.5218C>T p.(Arg1740Trp) and Group 2 consists of 3 individuals with heterozygous variant c.5219G>A p.(Arg1740Gln). The phenotype of Group 1 includes microcephaly, profound intellectual disability, congenital anomalies affecting several organ systems, and similar facial features. Individuals in Group 2 had moderate to severe intellectual disability, low normal head circumference, and absence of additional major congenital anomalies. While LLS is likely due to loss of function of SETD2, the clinical features seen in individuals with variants affecting codon 1740 are more severe suggesting an alternative mechanism, such as gain of function, effects on epigenetic regulation, or posttranslational modification of the cytoskeleton. Our report is a prime example of different mutations in the same gene causing diverging phenotypes and the features observed in Group 1 suggest a new clinically recognizable syndrome uniquely associated with the heterozygous variant c.5218C>T p.(Arg1740Trp) in SETD2.

Rapid identification of species, sex and maturity by mass spectrometric analysis of animal faeces.

BACKGROUND: We describe a new approach to the recovery of information from faecal samples, based on the analysis of the molecular signature generated by rapid evaporative ionisation mass spectrometry (REIMS). RESULTS: Faecal pellets from five different rodent species were analysed by REIMS, and complex mass spectra were acquired rapidly (typically a few seconds per sample). The uninterpreted mass spectra (signatures) were then used to seed linear discriminant analysis and classification models based on random forests. It was possible to classify each species of origin with a high rate of accuracy, whether faeces were from animals maintained under standard laboratory conditions or wild-caught. REIMS signatures were stable to prior storage of the faecal material under a range of different conditions and were not altered rapidly or radically by changes in diet. Further, within species, REIMS signatures could be used to discriminate faeces from adult versus juvenile mice, male versus female mice and those from three different laboratory strains. CONCLUSIONS: REIMS offers a completely novel method for the rapid analysis of faecal samples, extending faecal analysis (previously focused on DNA) to an assessment of phenotype, and has considerable potential as a new tool in the armamentarium of the field biologist.

Structural analysis of pathogenic mutations in the DYRK1A gene in patients with developmental disorders.

Haploinsufficiency in DYRK1A is associated with a recognizable developmental syndrome, though the mechanism of action of pathogenic missense mutations is currently unclear. Here we present 19 de novo mutations in this gene, including five missense mutations, identified by the Deciphering Developmental Disorder study. Protein structural analysis reveals that the missense mutations are either close to the ATP or peptide binding-sites within the kinase domain, or are important for protein stability, suggesting they lead to a loss of the protein's function mechanism. Furthermore, there is some correlation between the magnitude of the change and the severity of the resultant phenotype. A comparison of the distribution of the pathogenic mutations along the length of DYRK1A with that of natural variants, as found in the ExAC database, confirms that mutations in the N-terminal end of the kinase domain are more disruptive of protein function. In particular, pathogenic mutations occur in significantly closer proximity to the ATP and the substrate peptide than the natural variants. Overall, we suggest that de novo dominant mutations in DYRK1A account for nearly 0.5% of severe developmental disorders due to substantially reduced kinase function.

BCL11A Haploinsufficiency Causes an Intellectual Disability Syndrome and Dysregulates Transcription.

Intellectual disability (ID) is a common condition with considerable genetic heterogeneity. Next-generation sequencing of large cohorts has identified an increasing number of genes implicated in ID, but their roles in neurodevelopment remain largely unexplored. Here we report an ID syndrome caused by de novo heterozygous missense, nonsense, and frameshift mutations in BCL11A, encoding a transcription factor that is a putative member of the BAF swi/snf chromatin-remodeling complex. Using a comprehensive integrated approach to ID disease modeling, involving human cellular analyses coupled to mouse behavioral, neuroanatomical, and molecular phenotyping, we provide multiple lines of functional evidence for phenotypic effects. The etiological missense variants cluster in the amino-terminal region of human BCL11A, and we demonstrate that they all disrupt its localization, dimerization, and transcriptional regulatory activity, consistent with a loss of function. We show that Bcl11a haploinsufficiency in mice causes impaired cognition, abnormal social behavior, and microcephaly in accordance with the human phenotype. Furthermore, we identify shared aberrant transcriptional profiles in the cortex and hippocampus of these mouse models. Thus, our work implicates BCL11A haploinsufficiency in neurodevelopmental disorders and defines additional targets regulated by this gene, with broad relevance for our understanding of ID and related syndromes.

PIGT-CDG, a disorder of the glycosylphosphatidylinositol anchor: description of 13 novel patients and expansion of the clinical characteristics.

PURPOSE: To provide a detailed electroclinical description and expand the phenotype of PIGT-CDG, to perform genotype-phenotype correlation, and to investigate the onset and severity of the epilepsy associated with the different genetic subtypes of this rare disorder. Furthermore, to use computer-assisted facial gestalt analysis in PIGT-CDG and to the compare findings with other glycosylphosphatidylinositol (GPI) anchor deficiencies. METHODS: We evaluated 13 children from eight unrelated families with homozygous or compound heterozygous pathogenic variants in PIGT. RESULTS: All patients had hypotonia, severe developmental delay, and epilepsy. Epilepsy onset ranged from first day of life to two years of age. Severity of the seizure disorder varied from treatable seizures to severe neonatal onset epileptic encephalopathies. The facial gestalt of patients resembled that of previously published PIGT patients as they were closest to the center of the PIGT cluster in the clinical face phenotype space and were distinguishable from other gene-specific phenotypes. CONCLUSION: We expand our knowledge of PIGT. Our cases reaffirm that the use of genetic testing is essential for diagnosis in this group of disorders. Finally, we show that computer-assisted facial gestalt analysis accurately assigned PIGT cases to the multiple congenital anomalies-hypotonia-seizures syndrome phenotypic series advocating the additional use of next-generation phenotyping technology.