Determining the affinities of high-affinity antibodies using KinExA and surface plasmon resonance.

Accurate and efficient affinity measurement techniques are essential for the biophysical characterization of therapeutic monoclonal antibodies, one of the fastest growing drug classes. Surface plasmon resonance (SPR) is widely used for determining antibody affinity, but does not perform well with extremely high affinity (low picomolar to femtomolar range) molecules. In this study, we compare the SPR-based Carterra LSA and the kinetic exclusion assay (KinExA) for measuring the affinities of 48 antibodies generated against the SARS-CoV-2 receptor-binding domain. These data reveal that high-affinity antibodies can be generated straight from selections using high-quality in vitro library platforms with 54% correspondence between affinities measured using LSA and KinExA. Generally, where there was a 2-fold or greater difference between LSA and KinExA, KinExA reported that affinities were tighter. We highlight the differences between LSA and KinExA, identifying the benefits and pitfalls of each in terms of dynamic range and throughput. Furthermore, we demonstrate for the first time that single-point screening with KinExA can significantly improve throughput while maintaining a strong correlation with full binding curve equilibrium measurements, enabling the accurate rank-ordering of clones with exceptionally tight binding properties.

A Systematic Review of Evidence-Based Treatments for Adolescent and Adult Sexual Assault Victims.

BACKGROUND: Sexual assault (SA) is a serious crime that is a prevalent mental and public health problem. AIMS: Addressing the needs of SA victims and providing appropriate treatment are essential to reduce potential adverse short- and long-term outcomes. METHODS: Our team undertook an extensive systematic literature review (published between January 2006 and July 2021) to provide evidence-based mental health intervention recommendations for adolescent and adult victims of SA. Where SA-specific research was limited, the literature and clinical practice guidelines on treatments for trauma-induced post-traumatic stress disorder (PTSD) were reviewed to provide additional information to formulate recommendations. RESULTS: Findings strongly support several primary psychotherapy treatments: cognitive behavioral therapy, cognitive processing therapy, eye movement desensitization and reprocessing, narrative exposure therapy, and prolonged exposure therapy. Complementary (aerobic exercise, art, drama, and music therapy) and pharmacological treatments were explored. CONCLUSIONS: Mental health nurses who provide services for victims of SA can utilize this overview to guide recommendations for treatment of SA trauma and related PTSD symptoms to mitigate the short- and long-term negative impacts after a traumatic event. When victims of SA receive optimal mental health treatments, our communities benefit as victims heal and recover.

Treatment Delays in Nonsyndromic Craniosynostosis: A 30-Year Retrospective Case-Control Analysis of the Impact of Socioeconomic and Family Status on Access to Care.

BACKGROUND: Patients with nonsyndromic craniosynostosis (NSC) generally undergo corrective surgery before 1 year of age to the mitigate morbidities and risks of delayed repair. The cohort of patients who receive primary corrective surgery after 1 year and factors associated with their gaps to care is poorly characterized in literature. METHODS: A nested case-control study was conducted for NSC patients who underwent primary corrective surgery at our institution and affiliates between 1992 and 2022. Patients whose surgery occurred after 1 year of age were identified and matched 1:1 by surgical date to standard-care control subjects. Chart review was conducted to gather patient data regarding care timeline and sociodemographic characteristics. RESULTS: Odds of surgery after 1 year of age were increased in Black patients (odds ratio, 3.94; P < 0.001) and those insured by Medicaid (2.57, P = 0.018), with single caregivers (4.96, P = 0.002), and from lower-income areas (+1% per $1000 income decrease, P = 0.001). Delays associated with socioeconomic status primarily impacted timely access to a craniofacial provider, whereas caregiver status was associated with subspecialty level delays. These disparities were exacerbated in patients with sagittal and metopic synostosis, respectively. Patients with multisuture synostosis were susceptible to significant delays related to familial strain (foster status, insurer, and English proficiency). CONCLUSIONS: Patients from socioeconomically strained households face systemic barriers to accessing optimal NSC care; disparities may be exacerbated by the diagnostic/treatment complexities of specific types of craniosynostosis. Interventions at primary care and craniofacial specialist levels can decrease health care gaps and optimize outcomes for vulnerable patients.

Epidemiology and Etiology of Facial Injuries in Children.

Pediatric Trauma results in over 8 million emergency department visits and 11,000 deaths annually. Unintentional injuries continue to be the leader in morbidity and mortality in pediatric and adolescent populations in the United States. More than 10% of all visits to pediatric emergency rooms (ER) present with craniofacial injuries. The most common etiologies for facial injuries in children and adolescence are motor vehicle accidents, assault, accidental injuries, sports injuries, nonaccidental injuries (eg, child abuse) and penetrating injuries. In the United States, head trauma secondary to abuse is the leading cause of mortality among non-accidental trauma in this population.

Pediatric Orthognathic Surgery: A NSQIP-P Comparison of Peri-Operative Factors and Outcome Differences Between Cleft and Noncleft Patients.

OBJECTIVE: The present study aimed to investigate the risk factors, complication profiles, and clinical outcomes of cleft and noncleft patients undergoing single jaw (mandibular or LeFort 1) and bimaxillary (BSSO + LeFort 1). DESIGN: Retrospective Cross-sectional Study Setting: National Surgical Quality Improvement Program database 2018-2019. PATIENTS: Pediatric patients. INTERVENTIONS: Outcomes for mandibular, LeFort 1, and bimaxillary osteotomy were retrospectively evaluated for cleft and noncleft patients. MAIN OUTCOME MEASURES: Multivariate logistic regression was used to determine the odds of complications and length of stay for cleft and noncleft patients undergoing single jaw and double jaw surgery. RESULTS: 669 pediatric patient underwent orthognathic surgery in the study period; the majority received LF1 only (n = 385; 58.3%), followed by mandible only (n = 179; 27.1%), and bimaxillary (n = 105; 15.9%%). Cleft differences were present in 56% of LFI patients, 32% of mandibular patients, and 22% of bimaxillary patients. After multivariate adjustment, ASA class III was associated with nearly 400% increased odds of any complication including readmission and reoperation (OR = 5.99; CI [[1.54-23.32]], p < 0.01, and 65% increased LOS (ß-coefficient = 1.65, CI [1.37-1.99], p < 0.01). Presence of cleft was not significantly associated with odds of any complication (p = 0.69) nor increased LOS (p = 0.46) in this population. CONCLUSION: Complications remained low between surgery types among cleft and noncleft patients. The most significant risk factor in pediatric orthognathic surgery was not the presence of cleft but rather increased ASA class. Though common in patients seeking orthognathic surgery, cleft differences did not cause additional risk after adjustment for other variables.

New nurses apply only basic source evaluation criteria but realize their skills are lacking: More sophisticated approaches to teaching evaluation skills are required.

BACKGROUND: While information evaluation is an essential component of evidence based practice, it remains unclear how nurses perceive their own source evaluation skills and what evaluation criteria they typically apply. OBJECTIVES: This study aims to determine nurses' self-reported confidence in their evaluation skills and their actual source evaluation ability. The findings will guide information literacy instruction. METHODS: A questionnaire asked recently graduated nurses from four institutions in the Intermountain West (USA) to rate their confidence in evaluating information and to provide examples of evaluation criteria they typically applied. The quality of these criteria was rated by nursing librarians, then compared with reported confidence in evaluation, years employed as a nurse and highest degree level. RESULTS: While nurses' self-reported confidence levels about source evaluation largely matched their ability, their evaluation criteria showed a low level of sophistication and did not match the recommended criteria by professional organizations. Graduate education, not years of work experience, was predictive of the quality of criteria used by nurses, suggesting the importance of more instruction on source evaluation for nursing students. CONCLUSIONS: Nursing educators, including librarians, need to teach evaluation skills at the undergraduate level. Further investigation into building evaluation skills in nurses is warranted.

Case Management for Southwestern Hospice Organization: Process Improvement Plan.

Studies have shown that registered nurses are inadequately prepared to care for patients requiring hospice and palliative care. Reasons include inadequate curriculum, along with a lack of structured education related to hospice/palliative care and symptom management, which includes inadequate education on delivering home-based hospice/palliative case management. Challenges at the Southwestern Hospice Organization are consistent with industry standards, evidenced by a high level of afterhours triage phone calls related to ineffective case management setup and delivery upon patient admission to hospice service. Many of these triage inquires could be prevented with improved registered nurse case management education and subsequent execution. Through analyzing Southwestern Hospice Organization afterhours triage phone data, a deficiency in effective patient case management setup and delivery was defined. Best practices in hospice/palliative case management were then identified, and a quality improvement plan in the form of a nurse driven, hands-on, home hospice/palliative case management simulation was generated. Quality improvement for patient case management at the Southwestern Hospice Organization was the end goal.

Primary care management of chronic pelvic pain in women.

Chronic pelvic pain in women can arise from many causes and often results in significant declines in function and quality of life. A systematic approach for evaluating patients and initiating a management plan are recommended in the primary care setting. Comprehensive management strategies may include medication, pelvic physical therapy, and behavioral interventions.

Preclinical Development of EBI-005: An IL-1 Receptor-1 Inhibitor for the Topical Ocular Treatment of Ocular Surface Inflammatory Diseases.

OBJECTIVE: Topical interleukin (IL)-1 receptor (R)1 blockade is therapeutically active in reducing signs and symptoms of dry eye disease. Herein, we describe in vitro and in vivo nonclinical Investigational New Drug (IND)-enabling studies of EBI-005, a novel protein chimera of IL-1ß and IL-1 receptor antagonist (IL-1Ra or anakinra) that potently binds IL-1R1 and blocks signaling. These studies provide an assessment of receptor affinity, drug bioavailability, immunogenic response, safety, and tolerability in mice and rabbits. METHODS: In vitro and in silico along with Good Laboratory Practices (GLP) and non-GLP in vivo studies in mice and rabbits assessed the topical ocular and systemic immunogenicity and toxicology of EBI-005. Animals were treated with EBI-005 once daily subcutaneously or four times daily by topical ocular administration for up to 6 weeks (with 2-week recovery phase). RESULTS: EBI-005 has 500 times higher affinity than anakinra to IL-1R1. Predictive immunogenicity testing suggested that EBI-005 is not more immunogenic. Systemic bioavailability of EBI-005 is low (1.4% in mice and 0.2% in rabbits) after topical ocular administration. EBI-005 penetrated into the anterior ocular tissues within 15 min of topical ocular administration. However, it is low or undetectable after 4 hr and does not form a depot after repeated topical ocular administration. EBI-005 was safe and well tolerated, and exposure to drug was maintained despite an antidrug antibody response after systemic administration, based on IND-enabling toxicology and safety pharmacology studies. CONCLUSIONS: Ocular doses of EBI-005 at 50 mg/mL in mice and rabbits totaling 0.15 mg/eye in mice and 1.5 mg/eye in rabbits, administered 4 times daily, did not produce adverse effects, and demonstrated excellent bioavailability in target tissues with low systemic exposure. In addition, immunogenic response to the drug did not cause adverse effects or diminish the drug's activity in most cases. The results support drug administration of the highest anticipated human clinical study dose of a 20 mg/mL solution (40 µL 3 times daily in each eye).

The Gene Pool: The Ethics of Genetics in Primary Care.

AIM: The purpose of this integrative review is to critically analyze the research literature regarding ethical principles that surround the integration of genetics and genomics in primary care clinical practice. BACKGROUND: Advanced practice nurses (APRNs) play an important role in the provision of primary care services, in the areas of obstetrics, pediatrics, family practice, and internal medicine. Advances in genetic and genomic science are infiltrating these day-to-day health-care systems and becoming an integral part of health-care delivery. It is imperative for primary care providers to understand the ethical, legal, and social implications of genetics and genomics. METHODS: A comprehensive multistep search of CINAHL, MEDLINE, Academic Search Premier, PsycINFO, Web of Science, and Scopus databases was conducted to identify primary research articles published from 2003 to 2015 that evaluated ethical issues related to genetics and genomics in U. S. primary care practice. A sample of 26 primary research articles met the inclusion criteria. Whittemore and Knafl's (2005) revised framework for integrative reviews was used to guide the analysis and assess the quality of the studies. Key findings from the studies are discussed according to Beauchamp and Childress's (2009) ethical principles of autonomy, beneficence, nonmaleficence, and justice. RESULTS: Research conducted to date is mainly qualitative and descriptive and the analysis revealed several ethical challenges to implementing genetics and genomics in primary care settings. CONCLUSION: The review suggests that there are several implications for research, education, and the development of primary care practice that support APRNs delivering genetic and genomic care while incorporating knowledge of ethical principles. More research needs to be conducted that evaluates the actual genetic/genomic ethical issues encountered by primary care providers.

Patient Feedback on Orthodontic Therapist Student Outreach Training.

The introduction of orthodontic therapists as a new group of dental care professionals (DCPs) requires that their training in specialist orthodontic practices has provision for monitoring the level of supervision and clinical care provided by the students. The University of Warwick Diploma in Orthodontic Therapy programme has developed a patient questionnaire with the aim of assessing the patient's perception of the student. The observational questionnaire was designed to cover the four General Dental Council (GDC) domains and learning outcomes for orthodontic therapists, such that patient response could potentially provide valuable feedback to support the training programme. Each of the 10 students had 30 questionnaires to complete. The questionnaire was anonymous and it was designed to be suitable for both young patients and their carers to complete in less than five minutes. The response rate was very high, with 291 of the 300 questionnaires being completed. The level of co-operation may have been partly due to the ease of completion of the questionnaire, but might suggest the willingness to provide supportive feedback for the students. This study provided formative feedback to students' educational and clinical development without impacting on a student's clinical activity and will be used to develop further assessment tools.

Immediate Implant-based Prepectoral Breast Reconstruction Using a Vertical Incision.

BACKGROUND: Ideally, breast reconstruction is performed at the time of mastectomy in a single stage with minimal scarring. However, postoperative complications with direct-to-implant subpectoral reconstruction remain significant. These include asymmetry, flap necrosis, animation deformity, and discomfort. We report on a series of patients who have undergone immediate single-stage prepectoral, implant-based breast reconstruction with a smooth, adjustable saline implant covered with mesh/acellular dermal matrix for support using a vertical mastectomy incision. This technique, when combined with an adjustable implant, addresses the complications related to subpectoral implant placement of traditional expanders. Our follow-up time, 4.6 years (55 months), shows a low risk of implant loss and elimination of animation deformity while also providing patients with a safe and aesthetically pleasing result. METHODS: All patients who underwent immediate implant-based prepectoral breast reconstruction using a vertical mastectomy incision as a single-staged procedure were included. Charts were reviewed retrospectively. Adjustable smooth round saline implants and mesh/acellular dermal matrix were used for fixation in all cases. RESULTS: Thirty-one patients (62 breasts) underwent single-staged implant-based prepectoral breast reconstruction using a vertical mastectomy incision. Postoperative complications occurred in 9 patients, 6 of which were resolved with postoperative intervention while only 2 cases resulted in implant loss. CONCLUSIONS: There can be significant morbidity associated with traditional subpectoral implant-based breast reconstruction. As an alternative, the results of this study show that an immediate single-stage prepectoral breast reconstruction with a smooth saline adjustable implant, using a vertical incision, in conjunction with mesh/matrix support can be performed with excellent aesthetic outcomes and minimal complications.

Health literacy and obesity among native Hawaiian and Pacific Islanders in the United States.

OBJECTIVES: Our purpose was to describe relationships between demographic characteristics, body mass index (BMI), and health literacy among Native Hawaiians and other Pacific Islanders (NHPIs). DESIGN AND SAMPLE: In this cross-sectional survey, we interviewed 364 NHPI adults. MEASURES: We used Newest Vital Sign (NVS), a health literacy tool; measured heights and weights; and demographic questions. RESULTS: According to participants' NVS scores, 45.3% had at least a possibility of low health literacy. Lower NVS scores were associated with increased BMI (r = -0.12, p = .027) and increased age (r = -0.26, p < .001). Higher NVS scores were associated with higher incomes (r = 0.21, p = .001) and higher education (r = 0.27, p < .001). Women scored significantly better than men (t = -2.0, p = .05). Participants' NVS scores in Hawaii versus Utah were not significantly different (t = .26, p = .80). CONCLUSIONS: Pathways to health literacy are complex; however, age, income, education, and BMI explained a modest 19.95% of the combined variance in NVS scores. Public health nurses working to improve health literacy could include review of critical information on nutrition facts labels, frequently used calculations, and application of this information when making food choices.

The duplication 17p13.3 phenotype: analysis of 21 families delineates developmental, behavioral and brain abnormalities, and rare variant phenotypes.

Chromosome 17p13.3 is a gene rich region that when deleted is associated with the well-known Miller-Dieker syndrome. A recently described duplication syndrome involving this region has been associated with intellectual impairment, autism and occasional brain MRI abnormalities. We report 34 additional patients from 21 families to further delineate the clinical, neurological, behavioral, and brain imaging findings. We found a highly diverse phenotype with inter- and intrafamilial variability, especially in cognitive development. The most specific phenotype occurred in individuals with large duplications that include both the YWHAE and LIS1 genes. These patients had a relatively distinct facial phenotype and frequent structural brain abnormalities involving the corpus callosum, cerebellar vermis, and cranial base. Autism spectrum disorders were seen in a third of duplication probands, most commonly in those with duplications of YWHAE and flanking genes such as CRK. The typical neurobehavioral phenotype was usually seen in those with the larger duplications. We did not confirm the association of early overgrowth with involvement of YWHAE and CRK, or growth failure with duplications of LIS1. Older patients were often overweight. Three variant phenotypes included cleft lip/palate (CLP), split hand/foot with long bone deficiency (SHFLD), and a connective tissue phenotype resembling Marfan syndrome. The duplications in patients with clefts appear to disrupt ABR, while the SHFLD phenotype was associated with duplication of BHLHA9 as noted in two recent reports. The connective tissue phenotype did not have a convincing critical region. Our experience with this large cohort expands knowledge of this diverse duplication syndrome.

Orthopedic manifestations and implications for individuals with Costello syndrome.

Costello syndrome is a rare genetic condition caused by heterozygous alterations in HRAS and characterized by multi-system abnormalities. Individuals with Costello syndrome usually present with severe feeding difficulties in infancy, short stature, coarse facial features, increased tumor risks, cardiac and neurological complications, intellectual disability and orthopedic complications. This study further defines the orthopedic manifestations affecting individuals with Costello syndrome. We studied 43 participants and performed medical records review, clinical examinations and orthopedic inquiry forms. In 23 participants, hip and or spinal imaging assessments were completed. Serial radiographs were analyzed when available. A total of 25 orthopedic manifestations were identified. Ten manifestations were seen in the majority of the participants: hypotonia (87%), ligamentous laxity (85%), scoliosis (63%), kyphosis (58%), characteristic hand deformities (85%), ulnar deviation of the wrist (63%), elbow (55%) and shoulder contractures (65%), tight Achilles tendon (73%), and pes planus (53%). Other characteristics of special note were hip dysplasia (45%), foot deformities requiring surgical intervention (38%) and osteopenia/osteoporosis (47%). We also studied the development of the hips and spine. Uni- or bilateral hip dysplasia was congenital in some, while it developed throughout childhood in others. Spinal involvement included scoliosis, kyphosis, lordosis, and curvature reversal (thoracic lordosis and lumbar kyphosis). Based on these findings, we recommend routine referral to an orthopedic surgeon as well as instituting screening protocols for hips and spine for individuals with Costello syndrome.

Assessing genotype-phenotype correlation in Costello syndrome using a severity score.

PURPOSE: Costello syndrome, a rare genetic disorder with multisystemic involvement, is caused by germline HRAS mutations. Because several different missense mutations have been reported, a severity scoring system was developed to assess a possible genotype-phenotype correlation. METHODS: Records of 78 individuals with Costello syndrome were scored in early childhood, childhood, and young adulthood by a reviewer blinded to the individuals' specific mutations. These scores were based on certain medically relevant feeding, neurologic, orthopedic, endocrine, cardiac, malignancy, and mortality manifestations. Individuals' severity scores were then grouped by the particular HRAS mutation. The mixed-model approach for repeated-measures analysis of variance with unstructured within-subject correlation, pairwise comparisons, and contrast were used to determine whether the severity scores differed by mutation. RESULTS: Although the sample size was small, individuals with the p.G12A or p.G12C HRAS change were more severely affected than those with other HRAS mutations. Regardless of the mutation, severity did not increase significantly over time. CONCLUSION: Despite its limitations, including the small number of individuals with rare mutations and possibly incomplete medical records, this work providing the first quantitative assessment of phenotypic severity in a Costello syndrome cohort supports a medically relevant genotype-phenotype correlation.

Exonic deletions in AUTS2 cause a syndromic form of intellectual disability and suggest a critical role for the C terminus.

Genomic rearrangements involving AUTS2 (7q11.22) are associated with autism and intellectual disability (ID), although evidence for causality is limited. By combining the results of diagnostic testing of 49,684 individuals, we identified 24 microdeletions that affect at least one exon of AUTS2, as well as one translocation and one inversion each with a breakpoint within the AUTS2 locus. Comparison of 17 well-characterized individuals enabled identification of a variable syndromic phenotype including ID, autism, short stature, microcephaly, cerebral palsy, and facial dysmorphisms. The dysmorphic features were more pronounced in persons with 3'AUTS2 deletions. This part of the gene is shown to encode a C-terminal isoform (with an alternative transcription start site) expressed in the human brain. Consistent with our genetic data, suppression of auts2 in zebrafish embryos caused microcephaly that could be rescued by either the full-length or the C-terminal isoform of AUTS2. Our observations demonstrate a causal role of AUTS2 in neurocognitive disorders, establish a hitherto unappreciated syndromic phenotype at this locus, and show how transcriptional complexity can underpin human pathology. The zebrafish model provides a valuable tool for investigating the etiology of AUTS2 syndrome and facilitating gene-function analysis in the future.

Cardiac anomalies in Axenfeld-Rieger syndrome due to a novel FOXC1 mutation.

Axenfeld-Rieger syndrome (ARS) is an autosomal dominant condition characterized by ophthalmologic anterior segment abnormalities and extraocular findings including dental anomalies and redundant periumbilical skin. Intragenic mutations in the homeobox gene PITX2 or the transcription factor encoding FOXC1 were identified, and genomic rearrangements encompassing either gene also cause ARS. A molecular etiology is identified in 40-60%. Extraocular anomalies occur more often with intragenic PITX2 than FOXC1 mutations. We report on a patient with infantile glaucoma presenting at age 21 months with congestive heart failure due to a dysplastic arcade mitral valve necessitating valve replacement, and mildly hypoplastic left ventricular outflow tract and aortic arch. Family history included early onset glaucoma in four relatives; congenital hip dysplasia requiring surgery in three; and an atrial septal defect in the affected maternal grandmother. Despite the absence of dental or umbilical abnormalities, anterior chamber abnormalities consistent with ARS were present in affected individuals. Molecular testing revealed a novel FOXC1 mutation (c.508C>T; p.Arg170Trp) in the proband and his affected mother; other family members were unavailable. A literature review revealed four reports of congenital heart disease associated with intragenic FOXC1 mutations, and none with intragenic PITX2 mutations. Previously, mouse studies showed Foxc1 (Mf1) expression in the developing valves and atrial septum, supporting a causal relationship of FOXC1 mutations for valvar anomalies and ASD. Hip dysplasia in three family members suggests a role for FOXC1 in the femoral head dysplasia of de Hauwere syndrome with 6p25 deletions. Further reports of clinical and molecular diagnoses will clarify genotype-phenotype correlation.

Normative growth charts for individuals with Costello syndrome.

Costello syndrome is a rare condition due to heterozygous germline mutations in the proto-oncogene HRAS. It affects multiple organ systems and includes severe failure-to-thrive, short stature, and macrocephaly. The goal of this study was to develop Costello syndrome-specific growth curves. We collected height, weight, and head circumference (OFC) measurements from 94 individuals (45 males and 49 females). Their HRAS mutation spectrum reflects previously published cohorts, with p.G12S in 77.7%. Participants received medical care, therefore our data does not reflect natural history per se, but rather growth with nutritional support. Due to limited cohort size, we analyzed data from males and females together. Weight-for-age data included 417 separate measurements from 80 individuals age 0-36 months, and 585 measurements from 82 individuals for age 0-10 years. Height-for-age data were derived from 391 measurements from 77 individuals age 0-36 months, and 591 measurements from 90 individuals age 0-10 years. Measurements obtained after growth hormone exposure in 15 individuals were excluded in this analysis. The OFC curve was derived from 221 measurements from 55 individuals age 0-36 months. Centiles (5th, 50th, and 95th) were estimated across the age continuum for each growth parameter, and compared to gender-specific curves for average stature individuals. The resulting curves demonstrate very slow weight gain in the first 2 years. Short stature is seen in many, but after age 4 years the 95th centile for height falls within the low normal range for average stature children. Head circumference curves largely overlap those for average stature, reflecting relative macrocephaly.

A novel HRAS substitution (c.266C>G; p.S89C) resulting in decreased downstream signaling suggests a new dimension of RAS pathway dysregulation in human development.

Costello syndrome is caused by HRAS germline mutations affecting Gly(12) or Gly(13) in >90% of cases and these are associated with a relatively homogeneous phenotype. Rarer mutations in other HRAS codons were reported in patients with an attenuated or mild phenotype. Disease-associated HRAS missense mutations result in constitutive HRAS activation and increased RAF-MEK-ERK and PI3K-AKT signal flow. Here we report on a novel heterozygous HRAS germline alteration, c.266C>G (p.S89C), in a girl presenting with severe fetal hydrops and pleural effusion, followed by a more benign postnatal course. A sibling with the same mutation and fetal polyhydramnios showed a Dandy-Walker malformation; his postnatal course was complicated by severe feeding difficulties. Their apparently asymptomatic father is heterozygous for the c.266C>G change. By functional analyses we identified reduced levels of active HRAS(S89C) and diminished MEK, ERK and AKT phosphorylation in cells overexpressing HRAS(S89C) , which represent novel consequences of disease-associated HRAS mutations. Given our patients' difficult neonatal course and presence of this change in their asymptomatic father, we hypothesize that its harmful consequences may be time limited, with the late fetal stage being most sensitive. Alternatively, the phenotype may develop only in the presence of an additional as-yet-unknown genetic modifier. While the pathogenicity of the HRAS c.266C>G change remains unproven, our data may illustrate wide functional and phenotypic variability of germline HRAS mutations.