FOXP1 Haploinsufficiency Contributes to the Development of Congenital Diaphragmatic Hernia.

FOXP1 encodes a transcription factor involved in tissue regulation and cell-type-specific functions. Haploinsufficiency of FOXP1 is associated with a neurodevelopmental disorder: autosomal dominant mental retardation with language impairment with or without autistic features. More recently, heterozygous FOXP1 variants have also been shown to cause a variety of structural birth defects including central nervous system (CNS) anomalies, congenital heart defects, congenital anomalies of the kidney and urinary tract, cryptorchidism, and hypospadias. In this report, we present a previously unpublished case of an individual with congenital diaphragmatic hernia (CDH) who carries an approximately 3.8 Mb deletion. Based on this deletion, and deletions previously reported in two other individuals with CDH, we define a CDH critical region on chromosome 3p13 that includes FOXP1 and four other protein-coding genes. We also provide detailed clinical descriptions of two previously reported individuals with CDH who carry de novo, pathogenic variants in FOXP1 that are predicted to trigger nonsense-mediated mRNA decay. A subset of individuals with putatively deleterious FOXP4 variants has also been shown to develop CDH. Since FOXP proteins function as homo- or heterodimers and the homologs of FOXP1 and FOXP4 are expressed at the same time points in the embryonic mouse diaphragm, they may function together as a dimer, or in parallel as homodimers, to regulate gene expression during diaphragm development. Not all individuals with heterozygous, loss-of-function changes in FOXP1 develop CDH. Hence, we conclude that FOXP1 acts as a susceptibility factor that contributes to the development of CDH in conjunction with other genetic, epigenetic, environmental, and/or stochastic factors.

Expanding the clinical spectrum of biglycan-related Meester-Loeys syndrome.

Pathogenic loss-of-function variants in BGN, an X-linked gene encoding biglycan, are associated with Meester-Loeys syndrome (MRLS), a thoracic aortic aneurysm/dissection syndrome. Since the initial publication of five probands in 2017, we have considerably expanded our MRLS cohort to a total of 18 probands (16 males and 2 females). Segregation analyses identified 36 additional BGN variant-harboring family members (9 males and 27 females). The identified BGN variants were shown to lead to loss-of-function by cDNA and Western Blot analyses of skin fibroblasts or were strongly predicted to lead to loss-of-function based on the nature of the variant. No (likely) pathogenic missense variants without additional (predicted) splice effects were identified. Interestingly, a male proband with a deletion spanning the coding sequence of BGN and the 5' untranslated region of the downstream gene (ATP2B3) presented with a more severe skeletal phenotype. This may possibly be explained by expressional activation of the downstream ATPase ATP2B3 (normally repressed in skin fibroblasts) driven by the remnant BGN promotor. This study highlights that aneurysms and dissections in MRLS extend beyond the thoracic aorta, affecting the entire arterial tree, and cardiovascular symptoms may coincide with non-specific connective tissue features. Furthermore, the clinical presentation is more severe and penetrant in males compared to females. Extensive analysis at RNA, cDNA, and/or protein level is recommended to prove a loss-of-function effect before determining the pathogenicity of identified BGN missense and non-canonical splice variants. In conclusion, distinct mechanisms may underlie the wide phenotypic spectrum of MRLS patients carrying loss-of-function variants in BGN.

PhenoID, a language model normalizer of physical examinations from genetics clinical notes.

Background: Phenotypes identified during dysmorphology physical examinations are critical to genetic diagnosis and nearly universally documented as free-text in the electronic health record (EHR). Variation in how phenotypes are recorded in free-text makes large-scale computational analysis extremely challenging. Existing natural language processing (NLP) approaches to address phenotype extraction are trained largely on the biomedical literature or on case vignettes rather than actual EHR data. Methods: We implemented a tailored system at the Children's Hospital of Philadelpia that allows clinicians to document dysmorphology physical exam findings. From the underlying data, we manually annotated a corpus of 3136 organ system observations using the Human Phenotype Ontology (HPO). We provide this corpus publicly. We trained a transformer based NLP system to identify HPO terms from exam observations. The pipeline includes an extractor, which identifies tokens in the sentence expected to contain an HPO term, and a normalizer, which uses those tokens together with the original observation to determine the specific term mentioned. Findings: We find that our labeler and normalizer NLP pipeline, which we call PhenoID, achieves state-of-the-art performance for the dysmorphology physical exam phenotype extraction task. PhenoID's performance on the test set was 0.717, compared to the nearest baseline system (Pheno-Tagger) performance of 0.633. An analysis of our system's normalization errors shows possible imperfections in the HPO terminology itself but also reveals a lack of semantic understanding by our transformer models. Interpretation: Transformers-based NLP models are a promising approach to genetic phenotype extraction and, with recent development of larger pre-trained causal language models, may improve semantic understanding in the future. We believe our results also have direct applicability to more general extraction of medical signs and symptoms. Funding: US National Institutes of Health.

Clinical decision support with a comprehensive in-EHR patient tracking system improves genetic testing follow up.

OBJECTIVE: We sought to develop and evaluate an electronic health record (EHR) genetic testing tracking system to address the barriers and limitations of existing spreadsheet-based workarounds. MATERIALS AND METHODS: We evaluated the spreadsheet-based system using mixed effects logistic regression to identify factors associated with delayed follow up. These factors informed the design of an EHR-integrated genetic testing tracking system. After deployment, we assessed the system in 2 ways. We analyzed EHR access logs and note data to assess patient outcomes and performed semistructured interviews with users to identify impact of the system on work. RESULTS: We found that patient-reported race was a significant predictor of documented genetic testing follow up, indicating a possible inequity in care. We implemented a CDS system including a patient data capture form and management dashboard to facilitate important care tasks. The system significantly sped review of results and significantly increased documentation of follow-up recommendations. Interviews with key system users identified a range of sociotechnical factors (ie, tools, tasks, collaboration) that contribute to safer and more efficient care. DISCUSSION: Our new tracking system ended decades of workarounds for identifying and communicating test results and improved clinical workflows. Interview participants related that the system decreased cognitive and time burden which allowed them to focus on direct patient interaction. CONCLUSION: By assembling a multidisciplinary team, we designed a novel patient tracking system that improves genetic testing follow up. Similar approaches may be effective in other clinical settings.

Platelet findings in 22q11.2 deletion syndrome correlate with disease manifestations but do not correlate with GPIb surface expression.

Prior studies have demonstrated that patients with chromosome 22q11.2 deletion syndrome (22q11.2DS) have lower platelet counts (PC) compared to non-deleted populations. They also have an increased mean platelet volume. The mechanism for this has been postulated to be haploinsufficiency of the GPIBB gene. We examined platelet parameters, deletion size and factors known to influence counts, including status of thyroid hormone and congenital heart disease (CHD), in a population of 825 patients with 22q11.2DS. We also measured surface expression of GPIB-IX complex by flow cytometry. The major determinant of PC was deletion status of GP1BB, regardless of surface expression or other factors. Patients with nested distal chromosome 22q11.2 deletions (those with GP1BB present) had higher PCs than those with proximal deletions where GP1BB is deleted. Patients with 22q11.2DS also demonstrated an accelerated PC decrease with age, occurring in childhood. These data demonstrate that genes within the proximal deletion segment drive PC differences in 22q11.2DS and suggest that PC reference ranges may need to be adjusted for age and deletion size in 22q11.2DS populations. Bleeding did not correlate with either platelet count or GPIb expression. Further studies into drivers of expression of GPIb and associations with severe thrombocytopenia and immune thrombocytopenia are needed to inform clinical care.

Malate dehydrogenase 2 deficiency is an emerging cause of pediatric epileptic encephalopathy with a recognizable biochemical signature.

Malate dehydrogenases (MDH) serve a critical role in maintaining equilibrium of the NAD+/NADH ratio between the mitochondria and cytosol through the catalysis of the oxidation of L-malate to oxaloacetate in a reversible, NADH-dependent manner. MDH2 encodes the mitochondrial isoform, which is integral to the tricarboxylic acid cycle and thus energy homeostasis. Recently, five patients harboring compound heterozygous MDH2 variants have been described, three with early-onset epileptic encephalopathy, one with a stroke-like episode, and one with dilated cardiomyopathy. Here, we describe an additional seven patients with biallelic variants in MDH2, the largest and most neurodevelopmentally and ethnically diverse cohort to-date, including homozygous variants, a sibling pair, non-European patients, and an adult. From these patients, we learn that MDH2 deficiency results in a biochemical signature including elevations of plasma lactate and the lactate:pyruvate ratio with urinary excretion of malate. It also results in a recognizable constellation of neuroimaging findings of anterior-predominant cerebral atrophy, subependymal cysts with ventricular septations. We also recognize MDH2 deficiency as a cause of Leigh syndrome. Taken with existing patient reports, we conclude that MDH2 deficiency is an emerging and likely under-recognized cause of infantile epileptic encephalopathy and provide a framework for medical evaluation of patients identified with biallelic MDH2 variants.

The experience of one pediatric geneticist with telemedicine-based clinical diagnosis.

Telemedicine has long been considered as an attractive alternative methodology in clinical genetics to improve patient access and convenience. Given the importance of the dysmorphology physical examination and anthropometric measurement in clinical genetics, many have wondered if lost information would hamper diagnosis. We previously addressed this question by analyzing thousands of diagnostic encounters in a single practice involving multiple practitioners and found no evidence for a difference in new molecular diagnosis rates. However, our previous study design resulted in variability in providers between in-person and telemedicine evaluation groups. To address this in our present study, we expanded our analysis to 1104 new patient evaluations seen by one highly experienced clinical geneticist across two 10-month periods before and after the start of the COVID-19 pandemic. Comparing patients seen in-person to those seen by telemedicine, we found significant differences in race and ethnicity, preferred language, and home zip code median income. The clinical geneticist intended to send more genetic testing for those patients seen by telemedicine, but due to issues with test authorization and sample collection, there was no difference in ultimate completion rate between groups. We found no significant difference in new molecular diagnosis rate. Overall, we find telemedicine to be an acceptable alternative to in-person evaluation for routine pediatric clinical genetics care.

Identifying phenotypic expansions for congenital diaphragmatic hernia plus (CDH+) using DECIPHER data.

Congenital diaphragmatic hernia (CDH) can occur in isolation or in conjunction with other birth defects (CDH+). A molecular etiology can only be identified in a subset of CDH cases. This is due, in part, to an incomplete understanding of the genes that contribute to diaphragm development. Here, we used clinical and molecular data from 36 individuals with CDH+ who are cataloged in the DECIPHER database to identify genes that may play a role in diaphragm development and to discover new phenotypic expansions. Among this group, we identified individuals who carried putatively deleterious sequence or copy number variants affecting CREBBP, SMARCA4, UBA2, and USP9X. The role of these genes in diaphragm development was supported by their expression in the developing mouse diaphragm, their similarity to known CDH genes using data from a previously published and validated machine learning algorithm, and/or the presence of CDH in other individuals with their associated genetic disorders. Our results demonstrate how data from DECIPHER, and other public databases, can be used to identify new phenotypic expansions and suggest that CREBBP, SMARCA4, UBA2, and USP9X play a role in diaphragm development.

Clinical Effectiveness of Telemedicine-Based Pediatric Genetics Care.

BACKGROUND AND OBJECTIVES: Telemedicine may increase access to medical genetics care. However, in the pediatric setting, how telemedicine may affect the diagnostic rate is unknown, partially because of the perceived importance of the dysmorphology physical examination. We studied the clinical effectiveness of telemedicine for patients with suspected or confirmed genetic conditions. METHODS: We conducted a retrospective cohort study of outpatient encounters before and after the widespread implementation of telemedicine (N = 5854). Visit types, diagnoses, patient demographic characteristics, and laboratory data were acquired from the electronic health record. Patient satisfaction was assessed through survey responses. New molecular diagnosis was the primary end point. RESULTS: Patients seen by telemedicine were more likely to report non-Hispanic White ancestry, prefer to speak English, live in zip codes with higher median incomes, and have commercial insurance (all P < .01). Genetic testing was recommended for more patients evaluated by telemedicine than in person (79.5% vs 70.9%; P < .001). Patients seen in person were more likely to have a sample collected, resulting in similar test completion rates (telemedicine, 51.2%; in person, 55.1%; P = .09). There was no significant difference in molecular diagnosis rate between visit modalities (telemedicine, 13.8%; in person, 12.4%; P = .40). CONCLUSIONS: Telemedicine and traditional in-person evaluation resulted in similar molecular diagnosis rates. However, improved methodologies for remote sample collection may be required. This study reveals the feasibility of telemedicine in a large academic medical genetics practice and is applicable to other pediatric specialties with perceived importance of physical examination.

Distinct immune trajectories in patients with chromosome 22q11.2 deletion syndrome and immune-mediated diseases.

BACKGROUND: Identification of biomarkers associated with immune-mediated diseases in 22q11.2 deletion syndrome is an evolving field. OBJECTIVES: We sought to use a carefully phenotyped cohort to study immune parameters associated with autoimmunity and atopy in 22q11.2 deletion syndrome to define biomarkers associated with immune-mediated disease in this syndrome. METHODS: Chart review validated autoimmune disease and atopic condition diagnoses. Laboratory data were extracted for each subcohort and plotted according to age. A random-effects model was used to define statistical significance. RESULTS: CD19, CD4, and CD4/45RA lymphocyte populations were not different from the general cohort for patients with atopic conditions. CD4/45RA T cells were significantly lower in the subjects with immune thrombocytopenia compared with the general cohort, and CD4 T-cell counts were lower in patients with autoimmune thyroid disease. CONCLUSIONS: The mechanisms of autoimmunity in cytopenias may be distinct from those of solid-organ autoimmunity in 22q11.2 deletion syndrome. This study identifies potential biomarkers for risk stratification among commonly obtained laboratory studies.

Clinical exome sequencing data reveal high diagnostic yields for congenital diaphragmatic hernia plus (CDH+) and new phenotypic expansions involving CDH.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a life-threatening birth defect that often co-occurs with non-hernia-related anomalies (CDH+). While copy number variant (CNV) analysis is often employed as a diagnostic test for CDH+, clinical exome sequencing (ES) has not been universally adopted. METHODS: We analysed a clinical database of ~12 000 test results to determine the diagnostic yields of ES in CDH+ and to identify new phenotypic expansions. RESULTS: Among the 76 cases with an indication of CDH+, a molecular diagnosis was made in 28 cases for a diagnostic yield of 37% (28/76). A provisional diagnosis was made in seven other cases (9%; 7/76). Four individuals had a diagnosis of Kabuki syndrome caused by frameshift variants in KMT2D. Putatively deleterious variants in ALG12 and EP300 were each found in two individuals, supporting their role in CDH development. We also identified individuals with de novo pathogenic variants in FOXP1 and SMARCA4, and compound heterozygous pathogenic variants in BRCA2. The role of these genes in CDH development is supported by the expression of their mouse homologs in the developing diaphragm, their high CDH-specific pathogenicity scores generated using a previously validated algorithm for genome-scale knowledge synthesis and previously published case reports. CONCLUSION: We conclude that ES should be ordered in cases of CDH+ when a specific diagnosis is not suspected and CNV analyses are negative. Our results also provide evidence in favour of phenotypic expansions involving CDH for genes associated with ALG12-congenital disorder of glycosylation, Rubinstein-Taybi syndrome, Fanconi anaemia, Coffin-Siris syndrome and FOXP1-related disorders.

Proposed criteria for nevoid basal cell carcinoma syndrome in children assessed using statistical optimization.

Nevoid basal cell carcinoma syndrome (NBCCS) is a tumor predisposition condition, the cardinal features of which emerge in adolescence or adulthood. Using statistical optimization, this study proposes NBCCS criteria with improved sensitivity in children less than 18 years of age. Earlier detection may lead to improved surveillance and prevention of sequelae. A survey eliciting medical history was completed by, or on behalf of, individuals with NBCCS. Based on these findings, criteria for suspicion of NBCCS in children were suggested using information from a Bernoulli naïve Bayes classifier relying on the human phenotype ontology. The sensitivity and specificity of the existing and proposed diagnostic criteria were also assessed. Participants (n = 48) reported their first signs of NBCCS appeared at a median age of 8 months, but by our retrospective analysis, they did not fulfill the current diagnostic criteria until a median age of 7 years. This study delineates the early-onset features of NBCCS and proposes criteria that should prompt consideration of NBCCS. Additionally, we demonstrate a method for quantitatively assessing the utility of diagnostic criteria for genetic disorders.

Provider Perspectives on the Impact of the COVID-19 Pandemic on Newborn Screening.

The onset of the COVID-19 pandemic caused significant changes in healthcare delivery. Telemedicine rapidly and unexpectedly became the primary vehicle for ambulatory management. As newborn screen (NBS) referrals require varying levels of acuity, whether telemedicine could be used as a safe and effective medium to return these results were unknown. We sent an online survey to metabolism providers internationally to investigate triage differences of abnormal NBS results during the COVID-19 pandemic. The survey compared personal practice for the periods of March-June 2019 and March-June 2020. Responses were received from 44 providers practicing in 8 countries. Nearly all (93%) practiced in areas of widespread SARS-COV-2 community transmission during spring 2020. There was a significant expansion of telemedicine use for NBS referrals at the onset of the COVID-19 pandemic (OR: 12, 95% CI: 3.66-39.3, p < 0.0001). Telehealth primarily replaced in-person ambulatory metabolism visits. The increased frequency of virtual care was similar across NBS analytes. Providers found telehealth for NBS referral equally efficacious to in-person care. Institutional patient surveys showed no difference in satisfaction with provider communication, provider empathy, or appointment logistics. Our survey was limited by unprecedented disruption in healthcare delivery, necessitating further validation of telegenetics for NBS in the post-pandemic era. Nevertheless, our findings demonstrate that telemedicine is potentially a viable and practical tool for triaging abnormal NBS results.

Expanding the genotypic and phenotypic spectrum in a diverse cohort of 104 individuals with Wiedemann-Steiner syndrome.

Wiedemann-Steiner syndrome (WSS) is an autosomal dominant disorder caused by monoallelic variants in KMT2A and characterized by intellectual disability and hypertrichosis. We performed a retrospective, multicenter, observational study of 104 individuals with WSS from five continents to characterize the clinical and molecular spectrum of WSS in diverse populations, to identify physical features that may be more prevalent in White versus Black Indigenous People of Color individuals, to delineate genotype-phenotype correlations, to define developmental milestones, to describe the syndrome through adulthood, and to examine clinicians' differential diagnoses. Sixty-nine of the 82 variants (84%) observed in the study were not previously reported in the literature. Common clinical features identified in the cohort included: developmental delay or intellectual disability (97%), constipation (63.8%), failure to thrive (67.7%), feeding difficulties (66.3%), hypertrichosis cubiti (57%), short stature (57.8%), and vertebral anomalies (46.9%). The median ages at walking and first words were 20 months and 18 months, respectively. Hypotonia was associated with loss of function (LoF) variants, and seizures were associated with non-LoF variants. This study identifies genotype-phenotype correlations as well as race-facial feature associations in an ethnically diverse cohort, and accurately defines developmental trajectories, medical comorbidities, and long-term outcomes in individuals with WSS.

Vertical transmission of a large calvarial ossification defect due to heterozygous variants of ALX4 and TWIST1.

ALX4 is a homeobox gene expressed in the mesenchyme of developing bone and is known to play an important role in the regulation of osteogenesis. Enlarged parietal foramina (EPF) is a phenotype of delayed intramembranous ossification of calvarial bones due to variants of ALX4. The contrasting phenotype of premature ossification of sutures is observed with heterozygous loss-of-function variants of TWIST1, which is an important regulator of osteoblast differentiation. Here, we describe an individual with a large cranium defect, with dominant transmission from the mother, both carrying disease causing heterozygous variants in ALX4 and TWIST1. The distinct phenotype of absent superior and posterior calvarium in the child and his mother was in sharp contrast to the other affected maternal relatives with a recognizable ALX4-related EPF phenotype. This report demonstrates comorbid disorders of Saethre-Chotzen syndrome and EPF in a mother and her child, resulting in severe skull defects reminiscent of calvarial abnormalities observed with bilallelic ALX4 variants. To our knowledge this is the first instance of ALX4 and TWIST1 variants acting synergistically to cause a unique phenotype influencing skull ossification.

What is new with 22q? An update from the 22q and You Center at the Children's Hospital of Philadelphia.

22q11.2 deletion syndrome (22q11.2DS) is a disorder caused by recurrent, chromosome-specific, low copy repeat (LCR)-mediated copy-number losses of chromosome 22q11. The Children's Hospital of Philadelphia has been involved in the clinical care of individuals with what is now known as 22q11.2DS since our initial report of the association with DiGeorge syndrome in 1982. We reviewed the medical records on our continuously growing longitudinal cohort of 1,421 patients with molecularly confirmed 22q11.2DS from 1992 to 2018. Most individuals are Caucasian and older than 8 years. The mean age at diagnosis was 3.9 years. The majority of patients (85%) had typical LCR22A-LCR22D deletions, and only 7% of these typical deletions were inherited from a parent harboring the deletion constitutionally. However, 6% of individuals harbored other nested deletions that would not be identified by traditional 22q11.2 FISH, thus requiring an orthogonal technology to diagnose. Major medical problems included immune dysfunction or allergies (77%), palatal abnormalities (67%), congenital heart disease (64%), gastrointestinal difficulties (65%), endocrine dysfunction (>50%), scoliosis (50%), renal anomalies (16%), and airway abnormalities. Median full-scale intelligence quotient was 76, with no significant difference between individuals with and without congenital heart disease or hypocalcemia. Characteristic dysmorphic facial features were present in most individuals, but dermatoglyphic patterns of our cohort are similar to normal controls. This is the largest longitudinal study of patients with 22q11.2DS, helping to further describe the condition and aid in diagnosis and management. Further surveillance will likely elucidate additional clinically relevant findings as they age.

Prioritization of Candidate Genes for Congenital Diaphragmatic Hernia in a Critical Region on Chromosome 4p16 using a Machine-Learning Algorithm.

Wolf-Hirschhorn syndrome (WHS) is caused by partial deletion of the short arm of chromosome 4 and is characterized by dysmorphic facies, congenital heart defects, intellectual/developmental disability, and increased risk for congenital diaphragmatic hernia (CDH). In this report, we describe a stillborn girl with WHS and a large CDH. A literature review revealed 15 cases of WHS with CDH, which overlap a 2.3-Mb CDH critical region. We applied a machine-learning algorithm that integrates large-scale genomic knowledge to genes within the 4p16.3 CDH critical region and identified FGFRL1 , CTBP1 , NSD2 , FGFR3 , CPLX1 , MAEA , CTBP1-AS2 , and ZNF141 as genes whose haploinsufficiency may contribute to the development of CDH.

Predicting human genes susceptible to genomic instability associated with Alu/Alu-mediated rearrangements.

Alu elements, the short interspersed element numbering more than 1 million copies per human genome, can mediate the formation of copy number variants (CNVs) between substrate pairs. These Alu/Alu-mediated rearrangements (AAMRs) can result in pathogenic variants that cause diseases. To investigate the impact of AAMR on gene variation and human health, we first characterized Alus that are involved in mediating CNVs (CNV-Alus) and observed that these Alus tend to be evolutionarily younger. We then computationally generated, with the assistance of a supercomputer, a test data set consisting of 78 million Alu pairs and predicted ∼18% of them are potentially susceptible to AAMR. We further determined the relative risk of AAMR in 12,074 OMIM genes using the count of predicted CNV-Alu pairs and experimentally validated the predictions with 89 samples selected by correlating predicted hotspots with a database of CNVs identified by clinical chromosomal microarrays (CMAs) on the genomes of approximately 54,000 subjects. We fine-mapped 47 duplications, 40 deletions, and two complex rearrangements and examined a total of 52 breakpoint junctions of simple CNVs. Overall, 94% of the candidate breakpoints were at least partially Alu mediated. We successfully predicted all (100%) of Alu pairs that mediated deletions (n = 21) and achieved an 87% positive predictive value overall when including AAMR-generated deletions and duplications. We provided a tool, AluAluCNVpredictor, for assessing AAMR hotspots and their role in human disease. These results demonstrate the utility of our predictive model and provide insights into the genomic features and molecular mechanisms underlying AAMR.

Isolation of Aggressive Behavior Mutants in Drosophila Using a Screen for Wing Damage.

Aggression is a complex social behavior that is widespread in nature. To date, only a limited number of genes that affect aggression have been identified, in large part because the complexity of the phenotype makes screening difficult and time-consuming regardless of the species that is studied. We discovered that aggressive group-housed Drosophila melanogaster males inflict damage on each other's wings, and show that wing damage negatively affects their ability to fly and mate. Using this wing-damage phenotype, we screened males from ∼1400 chemically mutagenized strains and found ∼40 mutant strains with substantial wing damage. Five of these mutants also had increased aggressive behavior. To identify the causal mutation in one of our top aggressive strains, we used whole-genome sequencing and genomic duplication rescue strategies. We identified a novel mutation in the voltage-gated potassium channel Shaker (Sh) and show that a nearby previously identified Sh mutation also results in increased aggression. This simple screen can be used to dissect the molecular mechanisms underlying aggression.