Sensory experiences questionnaire unravels differences in sensory profiles between MECP2-related disorders.

The methyl CpG-binding protein-2 (MECP2) gene is located on the Xq28 region. Loss of function mutations or increased copies of MECP2 result in Rett syndrome (RTT) and MECP2 duplication syndrome (MDS), respectively. Individuals with both disorders exhibit overlapping autism symptoms, yet few studies have dissected the differences between these gene dosage sensitive disorders. Further, research examining sensory processing patterns in persons with RTT and MDS is largely absent. Thus, the goal of this study was to analyze and compare sensory processing patterns in persons with RTT and MDS. Towards this goal, caregivers of 50 female individuals with RTT and 122 male individuals with MDS, between 1 and 46 years of age, completed a standardized measure of sensory processing, the Sensory Experiences Questionnaire. Patterns detected in both disorders were compared against each other and against normative values. We found sensory processing abnormalities for both hyper- and hypo-sensitivity in both groups. Interestingly, abnormalities in MDS were more pronounced compared with in RTT, particularly with items concerning hypersensitivity and sensory seeking, but not hyposensitivity. Individuals with MDS also exhibited greater sensory symptoms compared with RTT in the areas of tactile and vestibular sensory processing and for both social and nonsocial stimuli. This study provides a first description of sensory symptoms in individuals with RTT and individuals with MDS. Similar to other neurodevelopmental disorders, a variety of sensory processing abnormalities was found. These findings reveal a first insight into sensory processing abnormalities caused by a dosage sensitive gene and may ultimately help guide therapeutic approaches for these disorders.

Development and validation of parent-reported gastrointestinal health scale in MECP2 duplication syndrome.

BACKGROUND/AIMS: We aimed to develop a validated patient-reported Gastrointestinal Health Scale (GHS) specific to MECP2 Duplication Syndrome (MDS) to be used in clinical trials. METHODS: MDS parents completed a Gastrointestinal Health Questionnaire (GHQ) to investigate the most relevant and important items associated with gastrointestinal problems in MECP2-related disorders. Item reduction was executed according to EORTC guidelines. We performed reliability and validity studies for the finalized scale. RESULTS: A total of 106 surveys were eligible for item reduction and validation processes. The initial 55 items were reduced to 38 items based on parent responses, expert opinion, and initial confirmatory factor analysis (CFA). The final MDS-specific GHS included 38 items and 7 factors that underwent further reliability and validity assessments. The power of the study was at least 0.982. The Cronbach's alphas of the instruments were General Health: 0.799, Eating-Chewing-Swallowing: 0.809, Reflux: 0.794, Motility: 0.762, Mood: 0.906, Medication: 0.595, Parenting: 0.942 and all items together: 0.928. The correlation coefficient between total and individual item scores ranged from 0.215 to 0.730. Because of the ordinal nature of the variables, the diagonal weighted least squares estimation (DWLS) method was used to execute the CFA and Structural Equation Modeling. The GHS had excellent model fit with the acceptable range of fit indices values. CONCLUSIONS: We developed a parent-reported, reliable, and valid MDS-specific GHS. This scale can be utilized in clinical settings or as an outcome measure in translational and clinical research.

Generation of five induced pluripotent stem cell lines from patients with MECP2 Duplication Syndrome.

MECP2 Duplication Syndrome (MDS) is a rare, severe neurodevelopmental disorder arising from duplications in the Xq28 region containing the MECP2 gene that predominantly affects males. We generated five human induced pluripotent stem cell (iPSC) lines from the fibroblasts of individuals carrying between 0.355 and 11.2 Mb size duplications in the chromosomal locus containing MECP2. All lines underwent extensive testing to confirm MECP2 duplication and iPSC-related features such as morphology, pluripotency markers, and trilineage differentiation potential. These lines are a valuable resource for molecular and functional studies of MDS as well as screening for a variety of therapeutic approaches.

Top caregiver concerns in Rett syndrome and related disorders: data from the US natural history study.

OBJECTIVE: Recent advances in the understanding of neurodevelopmental disorders such as Rett syndrome (RTT) have enabled the discovery of novel therapeutic approaches that require formal clinical evaluation of efficacy. Clinical trial success depends on outcome measures that assess clinical features that are most impactful for affected individuals. To determine the top concerns in RTT and RTT-related disorders we asked caregivers to list the top caregiver concerns to guide the development and selection of appropriate clinical trial outcome measures for these disorders. METHODS: Caregivers of participants enrolled in the US Natural History Study of RTT and RTT-related disorders (n = 925) were asked to identify the top 3 concerning problems impacting the affected participant. We generated a weighted list of top caregiver concerns for each of the diagnostic categories and compared results between the disorders. Further, for classic RTT, caregiver concerns were analyzed by age, clinical severity, and common RTT-causing mutations in MECP2. RESULTS: The top caregiver concerns for classic RTT were effective communication, seizures, walking/balance issues, lack of hand use, and constipation. The frequency of the top caregiver concerns for classic RTT varied by age, clinical severity, and specific mutations, consistent with known variation in the frequency of clinical features across these domains. Caregivers of participants with increased seizure severity often ranked seizures as the first concern, whereas caregivers of participants without active seizures often ranked hand use or communication as the top concern. Comparison across disorders found commonalities in the top caregiver concerns between classic RTT, atypical RTT, MECP2 duplication syndrome, CDKL5 deficiency disorder, and FOXG1 syndrome; however, distinct differences in caregiver concerns between these disorders are consistent with the relative prevalence and impact of specific clinical features. CONCLUSION: The top caregiver concerns for individuals with RTT and RTT-related disorders reflect the impact of the primary clinical symptoms of these disorders. This work is critical in the development of meaningful therapies, as optimal therapy should address these concerns. Further, outcome measures to be utilized in clinical trials should assess these clinical issues identified as most concerning by caregivers.

The development, content and response process validation of a caregiver-reported severity measure for CDKL5 deficiency disorder.

BACKGROUND: CDKL5 Deficiency Disorder (CDD) is a severe X-linked developmental and epileptic encephalopathy. Existing developmental outcome measures have floor effects and cannot capture incremental changes in symptoms. We modified the caregiver portion of a CDD clinical severity assessment (CCSA) and assessed content and response-process validity. METHODS: We conducted cognitive interviews with 15 parent caregivers of 1-39-year-old children with CDD. Caregivers discussed their understanding and concerns regarding appropriateness of both questions and answer options. Item wording and questionnaire structure were adjusted iteratively to ensure questions were understood as intended. RESULTS: The CCSA was refined during three rounds of cognitive interviews into two measures: (1) the CDD Developmental Questionnaire - Caregiver (CDQ-Caregiver) focused on developmental skills, and (2) the CDD Clinical Severity Assessment - Caregiver (CCSA-Caregiver) focused on symptom severity. Branching logic was used to ensure questions were age and skill appropriate. Initial pilot data (n = 11) suggested no floor effects. CONCLUSIONS: This study modified the caregiver portion of the initial CCSA and provided evidence for its content and response process validity.

Exploring gastrointestinal health in MECP2 duplication syndrome.

BACKGROUND: MECP2 duplication syndrome (MDS) is a rare neurogenetic syndrome caused by duplications of MECP2 at the Xq28 region. Although constipation and gastrointestinal reflux are reported in MDS, a comprehensive characterization of gastrointestinal health has not been fully explored. METHODS: We conducted a parent survey to explore the characteristics of gastrointestinal health in individuals with MDS using a secure online registry and compared differences in gastrointestinal symptoms between individuals with MDS and those with Rett syndrome (RTT). KEY RESULTS: One hundred six surveys were analyzed. Symptoms commonly associated with constipation occurred in 72% to 89% of MDS individuals. Eleven percent of MDS individuals underwent surgery for complications associated with constipation. We observed a bimodal distribution for gastroesophageal reflux disease (GERD) and gastrostomy feeding, with higher prevalence in 0-3 and >12-year-old MDS individuals. Constipation and GERD were significantly more common, and gas bloating was significantly less common in MDS than in RTT. Biliary tract disease requiring surgery was an unrecognized problem in 5% of MDS individuals. We determined that gastrointestinal problems in MDS individuals contribute to caretaker burden. CONCLUSION AND INFERENCES: Our study is the first in-depth investigation that characterizes gastrointestinal health in MDS and enumerates differences in gastrointestinal symptoms between MDS and RTT. Strategies to reduce gastrointestinal symptoms will alleviate caregiver burden in MDS. Further studies are needed to examine the mechanisms that cause gastrointestinal problems in MDS.

Epileptic spasms in CDKL5 deficiency disorder: Delayed treatment and poor response to first-line therapies.

OBJECTIVE: We aimed to assess the treatment response of infantile-onset epileptic spasms (ES) in CDKL5 deficiency disorder (CDD) vs other etiologies. METHODS: We evaluated patients with ES from the CDKL5 Centers of Excellence and the National Infantile Spasms Consortium (NISC), with onset from 2 months to 2 years, treated with adrenocorticotropic hormone (ACTH), oral corticosteroids, vigabatrin, and/or the ketogenic diet. We excluded children with tuberous sclerosis complex, trisomy 21, or unknown etiology with normal development because of known differential treatment responses. We compared the two cohorts for time to treatment and ES remission at 14 days and 3 months. RESULTS: We evaluated 59 individuals with CDD (79% female, median ES onset 6 months) and 232 individuals from the NISC database (46% female, median onset 7 months). In the CDD cohort, seizures prior to ES were common (88%), and hypsarrhythmia and its variants were present at ES onset in 34%. Initial treatment with ACTH, oral corticosteroids, or vigabatrin started within 1 month of ES onset in 27 of 59 (46%) of the CDD cohort and 182 of 232 (78%) of the NISC cohort (p < .0001). Fourteen-day clinical remission of ES was lower for the CDD group (26%, 7/27) than for the NISC cohort (58%, 106/182, p = .0002). Sustained ES remission at 3 months occurred in 1 of 27 (4%) of CDD patients vs 96 of 182 (53%) of the NISC cohort (p < .0001). Comparable results were observed with longer lead time (≥1 month) or prior treatment. Ketogenic diet, used within 3 months of ES onset, resulted in ES remission at 1 month, sustained at 3 months, in at least 2 of 13 (15%) individuals with CDD. SIGNIFICANCE: Compared to the broad group of infants with ES, children with ES in the setting of CDD often experience longer lead time to treatment and respond poorly to standard treatments. Development of alternative treatments for ES in CDD is needed.

Top Caregiver Concerns in Rett syndrome and related disorders: data from the US Natural History Study.

Objective: Recent advances in the understanding of neurodevelopmental disorders such as Rett syndrome (RTT) has enabled development of novel therapeutic approaches that are currently undergoing clinical evaluation or are proposed to move into clinical development. Clinical trial success depends on outcome measures that assess clinical features that are most impactful for affected individuals. To determine the top concerns in RTT and RTT-related disorders we asked caregivers to list the top clinical concerns in order to gain information to guide the development and selection of outcome measures for future clinical trials. Methods: Caregivers of participants enrolled in the US Natural History Study of RTT and related disorders were asked to identify the top 3 concerning problems impacting the affected participant. We generated a weighted list of top caregiver concerns for each of the diagnostic categories and compared results between the disorders. Further, for Classic RTT, caregiver concerns were analyzed by age, clinical severity, and common RTT-causing mutations in MECP2. Results: The top caregiver concerns for Classic RTT were effective communication, seizures, walking/balance issues, lack of hand use, and constipation. The rank order of the frequency of the top caregiver concerns for Classic RTT varied by age, clinical severity, and specific mutations, consistent with known variation in the frequency of clinical features across these domains. The frequency of caregiver concern for seizures, hand use, and spoken language increased in relation to clinician assessed severity in these clinical domains, showing consistency between clinician assessments and caregiver concerns. Comparison across disorders found commonalities in the top caregiver concerns between Classic RTT, Atypical RTT, MECP2 Duplication Syndrome, CDKL5 Deficiency Disorder, and FOXG1 Syndrome; however, distinct differences in caregiver concerns between these disorders are consistent with the relative prevalence and impact of specific clinical features. Conclusion: The top caregiver concerns for individuals with RTT and the RTT-related disorders reflect the impact of the primary clinical symptoms of these disorders. This work is critical in the development of meaningful therapies, as optimal therapy should address these concerns. Further, outcome measures to be utilized in clinical trials should assess these clinical issues identified as most concerning by caregivers.

COVID-19 Induced Environments, Health-Related Quality of Life Outcomes and Problematic Behaviors: Evidence from Children with Syndromic Autism Spectrum Disorders.

Between July 2020 and January 2021, 230 principal caregivers completed a questionnaire to measure proxy-assessed health-related quality of life outcomes (HRQoL), behavioral outcomes in children with syndromic autism spectrum disorders and COVID-19 induced changes to lifestyle and environments. HRQoL and behavioral outcomes reported earlier during the pandemic were generally worse compared to those reported later. COVID-19 induced reduction to a caregiver's mental health appointments, and hours spent watching TV were associated with decreases in HRQoL and increased the likelihood of problematic behaviors. Increasing time outdoors and time away from digital devices were positively associated with HRQoL and behaviors and might protect children from COVID-19 induced restrictions.

Distribution of hand function by age in individuals with Rett syndrome.

Objective: To determine the longitudinal distribution of hand function skills in individuals with classic Rett Syndrome (RTT), an X-linked dominant neurodevelopmental disorder, and correlate with MECP2 variants. Method: We conducted a longitudinal study of 946 girls and young women with typical RTT seen between 2006 and 2021 in the US Natural History Study (NHS) featuring a structured clinical evaluation to assess the level of hand function skills. The specific focus in this study was to assess longitudinal variation of hand skills from age 2 through age 18 years in relation to specific MECP2 variant groups. Results: Following the initial regression period, hand function continues to decline across the age spectrum in individuals with RTT. Specific differences are noted with steeper declines in hand function among those with milder variants (Group A: R133C, R294X, R306C, and C-terminal truncations) compared to groups composed of individuals with more severe variants. Conclusions: These temporal variations in hand use represent specific considerations which could influence the design of clinical trials that test therapies aiming to ameliorate specific functional limitations in individuals with RTT. Furthermore, the distinct impact of specific MECP2 variants on clinical severity, especially related to hand use, should be considered in such interventional trials.

Resilience, and positive parenting in parents of children with syndromic autism and intellectual disability. Evidence from the impact of the COVID-19 pandemic on family's quality of life and parent-child relationships.

Family quality of life (FQoL) outcomes collected during the first year of COVID-19 has been combined with 2018 data to estimate the outbreak's impact on parental outcomes on a sample of 230 families with syndromic autistic children and those with intellectual disabilities (IDs). Despite challenges imposed by the COVID-19 outbreak, our study found that FQoL outcomes reported by participating parents during the first year of COVID-19 appears to be similar to ratings from a prepandemic study of families with the same conditions. Parents of children in our sample generally displayed a stable functioning trajectory as measured by the validated FQoL instrument. Across syndromic autistic groups considered, families reported that their relationships with their children were positive. Our findings provide evidence of families' resilience which might explain the presence of positive parent-child interactions during COVID-19. Exploring mechanisms which would explain how families with autistic and ID children confront, manage disruptive experiences, and buffer COVID-19 induced stress is a fruitful direction for future research.

Assessing the Burden on Caregivers of MECP2 Duplication Syndrome.

BACKGROUND: MECP2 duplication syndrome (MDS) is a rare neurogenetic disorder characterized by severe neurodevelopmental disorder, refractory epilepsy, recurrent infections, and functional gastrointestinal problems. Because of the significant clinical problems and lifelong disability of children with this disorder we hypothesized that the burden on parents/caregivers of these children is significant. However, there are no reports of the impact on caregivers of individuals with MDS. METHODS: We developed and validated a burden scale to investigate the challenges of caregivers of children and adults with MDS and identified factors contributing to the burden on caregivers. We developed a Health Insurance Portability and Accountability Act-compliant patient registry for families with MDS and delivered caregiver burden survey through the registry. RESULTS: Of 237 completed surveys, 101 were eligible for the study. We identified increased levels of self-perceived anxiety, depression, and emotional exhaustion in caregivers that correlated with higher burden scores. Epilepsy was the only clinical feature that caused a higher burden in caregivers of individuals with MDS. In addition, a higher burden was found in Hispanic caregivers. The duration of care negatively correlated with burden score. CONCLUSIONS: This is the first study to investigate the burden on caregivers of individuals with MDS and identify several factors contributing to increased burden. Addressing these concerns has the potential to improve the health of individuals with MDS and contribute to the well-being of their caretakers.

Exploring the characteristics and most bothersome symptoms in MECP2 duplication syndrome to pave the path toward developing parent-oriented outcome measures.

BACKGROUND: MECP2 Duplication Syndrome (MDS), resulting from the duplication of Xq28 region, including MECP2, is a rare disorder with a nascent understanding in clinical features and severity. Studies using antisense oligonucleotides revealed a broad phenotypic rescue in transgenic mice. With human clinical trials on the horizon, there is a need to develop clinical outcome measures for MDS. METHODS: We surveyed caregivers of MDS individuals to explore the frequency and severity of MDS clinical features, and identify the most meaningful symptoms/domains that need to be included in the outcome measure scales. RESULTS: A total of 101 responses were eligible for the survey. The top six most meaningful symptoms to caregivers in descending order included epilepsy, gross motor, fine motor, communication, infection, and constipation problems. Epilepsy was present in 58.4% of the subjects and 75% were drug-resistant, Furthermore, ~12% required intensive care unit (ICU) admission. Infections were present in 55% of the subjects, and one-fourth of them required ICU admission. Constipation was present in ~85% of the subjects and one-third required enemas/suppositories. CONCLUSION: Our study is one of the largest cohorts conducted on MDS individuals characterizing the frequency and severity of MDS symptoms. Additionally, these study results will contribute to establishing a foundation to develop parent-reported outcomes in MDS.

Characteristic behaviors associated with gait of individuals with Rett syndrome.

BACKGROUND: Individuals with Rett syndrome (RTT) exhibit impaired motor performance and gait performance, leading to decreased quality of life. Currently, there is no robust observational instrument to identify gait characteristics in RTT. Current scales are limited as individuals with intellectual disorders may be unable to understand instructions. Our primary purpose was to utilize video analysis to characterize the behaviors associated with walking in individuals with RTT and explore the relationship between behaviors during overground and during treadmill walking. METHODS: Fourteen independently ambulatory females with RTT were video-taped and observed during overground and treadmill walking. Their gait was codified into an observational checklist to reveal prominent features associated with gait in this population. RESULTS: Participants exhibited similar rates of freezing, veering, and hand stereotypies between overground and treadmill walking; however, freeze duration was shortened during treadmill walking. Toe walking was prominently exhibited during overground, but not treadmill walking. During both walking modes, participants required extensive external motivation to maintain their walking patterns. CONCLUSIONS: Results identify several gait characteristics observable during overground and treadmill walking. In general, participants behaved similarly during overground and treadmill walking. We conclude that both overground and treadmill walking are appropriate tools to evaluate gait in this population.Implications for rehabilitationLocomotor rehabilitation may increase the quantity of walking performed by the patients, which can alleviate negative effects of the sedentary lifestyle commonly observed in patients with Rett syndrome (RTT).Video analysis of natural walking can be an effective tool to characterize gait in patients with RTT which does not require particular instructions which may not be fully understood.Both overground and treadmill walking are appropriate means of evaluating gait in individuals with RTT.

Health-Related Quality of Life in Pediatric Patients with Syndromic Autism and their Caregivers.

Children with autism have a significantly lower quality of life compared with their neurotypical peers. While multiple studies have quantified the impact of autism on health-related quality of life (HRQoL) through standardized surveys such as the PedsQL, none have specifically investigated the impact of syndromic autism. Here we evaluate HRQoL in children diagnosed with three genetic disorders that strongly predispose to syndromic autism: Phelan-McDermid syndrome (PMD), Rett syndrome (RTT), and SYNGAP1-related intellectual disability (SYNGAP1-ID). We find the most severely impacted dimension is physical functioning. Strikingly, syndromic autism results in worse quality of life than other chronic disorders including idiopathic autism. This study demonstrates the utility of caregiver surveys in prioritizing phenotypes, which may be targeted as clinical endpoints for genetically defined ASDs.

Current neurologic treatment and emerging therapies in CDKL5 deficiency disorder.

BACKGROUND: CDKL5 deficiency disorder (CDD) is associated with refractory infantile onset epilepsy, global developmental delay, and variable features that include sleep, behavioral disturbances, and movement disorders. Current treatment is primarily symptom-based and informed by experience in caring for this population. METHODS: We describe medication and non-medication approaches to treatment of epilepsy and additional key neurologic symptoms (sleep disturbances, behavioral issues, movement disorders, and swallowing dysfunction) in a cohort of 177 individuals meeting criteria for CDD, 154 evaluated at 4 CDKL5 Centers of Excellence in the USA and 40 identified through the NIH Natural History Study of Rett and Related Disorders. RESULTS: The four most frequently prescribed anti-seizure medications were broad spectrum, prescribed in over 50% of individuals. While the goal was not to ascertain efficacy, we obtained data from 86 individuals regarding response to treatment, with 2-week response achieved in 14-48% and sustained 3-month response in 5-36%, of those with known response. Additional treatments for seizures included cannabis derivatives, tried in over one-third of individuals, and clinical trial medications. In combination with pharmacological treatment, 50% of individuals were treated with ketogenic diet for attempted seizure control. Surgical approaches included vagus nerve stimulators, functional hemispherectomy, and corpus callosotomy, but numbers were too limited to assess response. Nearly one-third of individuals received pharmacologic treatment for sleep disturbances, 13% for behavioral dysregulation and movement disorders, and 43% had gastrostomy tubes. CONCLUSIONS: Treatment for neurologic features of CDD is currently symptom-based and empiric rather than CDD-specific, though clinical trials for CDD are emerging. Epilepsy in this population is highly refractory, and no specific anti-seizure medication was associated with improved seizure control. Ketogenic diet is commonly used in patients with CDD. While behavioral interventions are commonly instituted, information on the use of medications for sleep, behavioral management, and movement disorders is sparse and would benefit from further characterization and optimization of treatment approaches. The heterogeneity in treatment approaches highlights the need for systematic review and guidelines for CDD. Additional disease-specific and disease-modifying treatments are in development.

Content Validation of Clinician-Reported Items for a Severity Measure for CDKL5 Deficiency Disorder.

CDKL5 deficiency disorder (CDD) results in early-onset seizures and severe developmental impairments. A CDD clinical severity assessment (CCSA) was previously developed with clinician and parent-report items to capture information on a range of domains. Consistent with US Food and Drug Administration (FDA) guidelines, content validation is the first step in evaluating the psychometric properties of an outcome measure. The aim of this study was to validate the content of the clinician-reported items in the CCSA (CCSA-Clinician). Eight neurologists leading the USA CDD Center of Excellence clinics were interviewed using the "think aloud" technique to critique 26 clinician-reported items. Common themes were aggregated, and a literature search of related assessments informed item modifications. The clinicians then participated in 2 consensus meetings to review themes and finalize the items. A consensus was achieved for the content of the CCSA-Clinician. Eight of the original items were omitted, 11 items were added, and the remaining 18 items were revised. The final 29 items were classified into 2 domains: functioning and neurologic impairments. This study enabled refinement of the CCSA-Clinician and provided evidence for its content validity. This preliminary validation is essential before field testing and further validation, in order to advance the instrument toward clinical trial readiness.

Inhibition of Elevated Ras-MAPK Signaling Normalizes Enhanced Motor Learning and Excessive Clustered Dendritic Spine Stabilization in the MECP2-Duplication Syndrome Mouse Model of Autism.

The inflexible repetitive behaviors and "insistence on sameness" seen in autism imply a defect in neural processes controlling the balance between stability and plasticity of synaptic connections in the brain. It has been proposed that abnormalities in the Ras-ERK/MAPK pathway, a key plasticity-related cell signaling pathway known to drive consolidation of clustered synaptic connections, underlie altered learning phenotypes in autism. However, a link between altered Ras-ERK signaling and clustered dendritic spine plasticity has yet to be explored in an autism animal model in vivo The formation and stabilization of dendritic spine clusters is abnormally increased in the MECP2-duplication syndrome mouse model of syndromic autism, suggesting that ERK signaling may be increased. Here, we show that the Ras-ERK pathway is indeed hyperactive following motor training in MECP2-duplication mouse motor cortex. Pharmacological inhibition of ERK signaling normalizes the excessive clustered spine stabilization and enhanced motor learning behavior in MECP2-duplication mice. We conclude that hyperactive ERK signaling may contribute to abnormal clustered dendritic spine consolidation and motor learning in this model of syndromic autism.

DoMY-Seq: A yeast two-hybrid-based technique for precision mapping of protein-protein interaction motifs.

Interactions between proteins are fundamental for every biological process and especially important in cell signaling pathways. Biochemical techniques that evaluate these protein-protein interactions (PPIs), such as in vitro pull downs and coimmunoprecipitations, have become popular in most laboratories and are essential to identify and validate novel protein binding partners. Most PPIs occur through small domains or motifs, which are challenging and laborious to map by using standard biochemical approaches because they generally require the cloning of several truncation mutants. Moreover, these classical methodologies provide limited resolution of the interacting interface. Here, we describe the development of an alternative technique to overcome these limitations termed "Protein Domain mapping using Yeast 2 Hybrid-Next Generation Sequencing" (DoMY-Seq), which leverages both yeast two-hybrid and next-generation sequencing techniques. In brief, our approach involves creating a library of fragments derived from an open reading frame of interest and enriching for the interacting fragments using a yeast two-hybrid reporter system. Next-generation sequencing is then subsequently employed to read and map the sequence of the interacting fragment, yielding a high-resolution plot of the binding interface. We optimized DoMY-Seq by taking advantage of the well-described and high-affinity interaction between KRAS and CRAF, and we provide high-resolution domain mapping on this and other protein-interacting pairs, including CRAF-MEK1, RIT1-RGL3, and p53-MDM2. Thus, DoMY-Seq provides an unbiased alternative method to rapidly identify the domains involved in PPIs by advancing the use of yeast two-hybrid technology.