The localization of PHRAGMOPLAST ORIENTING KINESIN1 at the division site depends on the microtubule-binding proteins TANGLED1 and AUXIN-INDUCED IN ROOT CULTURES9 in Arabidopsis.

Proper plant growth and development require spatial coordination of cell divisions. Two unrelated microtubule-binding proteins, TANGLED1 (TAN1) and AUXIN-INDUCED IN ROOT CULTURES9 (AIR9), are together required for normal growth and division plane orientation in Arabidopsis (Arabidopsis thaliana). The tan1 air9 double mutant has synthetic growth and division plane orientation defects, while single mutants lack obvious defects. Here we show that the division site-localized protein, PHRAGMOPLAST ORIENTING KINESIN1 (POK1), was aberrantly lost from the division site during metaphase and telophase in the tan1 air9 mutant. Since TAN1 and POK1 interact via the first 132 amino acids of TAN1 (TAN11-132), we assessed the localization and function of TAN11-132 in the tan1 air9 double mutant. TAN11-132 rescued tan1 air9 mutant phenotypes and localized to the division site during telophase. However, replacing six amino-acid residues within TAN11-132, which disrupted the POK1-TAN1 interaction in the yeast-two-hybrid system, caused loss of both rescue and division site localization of TAN11-132 in the tan1 air9 mutant. Full-length TAN1 with the same alanine substitutions had defects in phragmoplast guidance and reduced TAN1 and POK1 localization at the division site but rescued most tan1 air9 mutant phenotypes. Together, these data suggest that TAN1 and AIR9 are required for POK1 localization, and yet unknown proteins may stabilize TAN1-POK1 interactions.

Defects in division plane positioning in the root meristematic zone affect cell organization in the differentiation zone.

Cell-division-plane orientation is critical for plant and animal development and growth. TANGLED1 (TAN1) and AUXIN-INDUCED IN ROOT CULTURES 9 (AIR9) are division-site-localized microtubule-binding proteins required for division-plane positioning. The single mutants tan1 and air9 of Arabidopsis thaliana have minor or no noticeable phenotypes, but the tan1 air9 double mutant has synthetic phenotypes including stunted growth, misoriented divisions and aberrant cell-file rotation in the root differentiation zone. These data suggest that TAN1 plays a role in non-dividing cells. To determine whether TAN1 is required in elongating and differentiating cells in the tan1 air9 double mutant, we limited its expression to actively dividing cells using the G2/M-specific promoter of the syntaxin KNOLLE (pKN:TAN1-YFP). Unexpectedly, in addition to rescuing division-plane defects, expression of pKN:TAN1-YFP rescued root growth and cell file rotation defects in the root-differentiation zone in tan1 air9 double mutants. This suggests that defects that occur in the meristematic zone later affect the organization of elongating and differentiating cells.

The Impact of Social Media on College Mental Health During the COVID-19 Pandemic: a Multinational Review of the Existing Literature.

PURPOSE OF REVIEW: During the COVID-19 pandemic, both social media use and rates of anxiety and depression among college students have increased significantly. This begs the question, what is the relationship between social media use and college student mental health during the COVID-19 pandemic? RECENT FINDINGS: Prior studies have found mixed results regarding the relationship between social media use and college student mental health. This relationship has become increasingly complex during the COVID-19 pandemic. We found that excessive or problematic social media use during the COVID-19 pandemic was correlated with worse mental health outcomes that could be mitigated by dialectical thinking, optimism, mindfulness, and cognitive reappraisal. The COVID-19 pandemic acts as a moderator by strengthening the relationship between social media use and mental health. Future studies should consider the impact of social media on college student mental health and concentrate on intervention initiatives to ensure the psychological well-being of college students during a global pandemic outbreak.

Musculoskeletal Urgent Care Centers Restrict Access for Patients with Medicaid Insurance Based on Policy and Location.

BACKGROUND: As the urgent care landscape evolves, specialized musculoskeletal urgent care centers (MUCCs) are becoming more prevalent. MUCCs have been offered as a convenient, cost-effective option for timely acute orthopaedic care. However, a recent "secret-shopper" study on patient access to MUCCs in Connecticut demonstrated that patients with Medicaid had limited access to these orthopaedic-specific urgent care centers. To investigate how generalizable these regional findings are to the United States, we conducted a nationwide secret-shopper study of MUCCs to identify determinants of patient access. QUESTIONS/PURPOSES: (1) What proportion of MUCCs in the United States provide access for patients with Medicaid insurance? (2) What factors are associated with MUCCs providing access for patients with Medicaid insurance? (3) What barriers exist for patients seeking care at MUCCs? METHODS: An online search of all MUCCs across the United States was conducted in this cross-sectional study. Three separate search modalities were used to gather a complete list. Of the 565 identified, 558 were contacted by phone with investigators posing over the telephone as simulated patients seeking treatment for a sprained ankle. Thirty-nine percent (216 of 558) of centers were located in the South, 13% (71 of 558) in the West, 25% (138 of 558) in the Midwest, and 24% (133 of 558) in New England. This study was given an exemption waiver by our institution's IRB. MUCCs were contacted using a standardized script to assess acceptance of Medicaid insurance and identify barriers to care. Question 1 was answered through determining the percentage of MUCCs that accepted Medicaid insurance. Question 2 considered whether there was an association between Medicaid acceptance and factors such as Medicaid physician reimbursements or MUCC center type. Question 3 sought to characterize the prevalence of any other means of limiting access for Medicaid patients, including requiring a referral for a visit and disallowing continuity of care at that MUCC. RESULTS: Of the MUCCs contacted, 58% (323 of 558) accepted Medicaid insurance. In 16 states, the proportion of MUCCs that accepted Medicaid was equal to or less than 50%. In 22 states, all MUCCs surveyed accepted Medicaid insurance. Academic-affiliated MUCCs accepted Medicaid patients at a higher proportion than centers owned by private practices (odds ratio 14 [95% CI 4.2 to 44]; p < 0.001). States with higher Medicaid physician reimbursements saw proportional increases in the percentage of MUCCs that accepted Medicaid insurance under multivariable analysis (OR 36 [95% CI 14 to 99]; p < 0.001). Barriers to care for Medicaid patients characterized included location restriction and primary care physician referral requirements. CONCLUSION: It is clear that musculoskeletal urgent care at these centers is inaccessible to a large segment of the Medicaid-insured population. This inaccessibility seems to be related to state Medicaid physician fee schedules and a center's affiliation with a private orthopaedic practice, indicating how underlying financial pressures influence private practice policies. Ultimately, the refusal of Medicaid by MUCCs may lead to disparities in which patients with private insurance are cared for at MUCCs, while those with Medicaid may experience delays in care. Going forward, there are three main options to tackle this issue: increasing Medicaid physician reimbursement to provide a financial incentive, establishing stricter standards for MUCCs to operate at the state level, or streamlining administration to reduce costs overall. Further research will be necessary to evaluate which policy intervention will be most effective. LEVEL OF EVIDENCE: Level II, prognostic study.

Urgent Care Centers Delay Emergent Surgical Care Based on Patient Insurance Status in The United States.

OBJECTIVE: Patients may call urgent care centers (UCCs) with urgent surgical conditions but may not be properly referred to a higher level of care. This study aims to characterize how UCCs manage Medicaid and privately insured patients who present with an emergent condition. METHODS: Using a standardized script, we called 1245 randomly selected UCCs in 50 states on 2 occasions. Investigators posed as either a Medicaid or a privately-insured patient with symptoms of an incarcerated inguinal hernia. Rates of direct emergency department (ED) referral were compared between insurance types. RESULTS: A total of 1223 (98.2%) UCCs accepted private insurance and 981 (78.8%) accepted Medicaid. At the 971 (78.0%) UCCs that accepted both insurance types, direct-to-ED referral rates for private and Medicaid patients were 27.9% and 33.8%, respectively. Medicaid patients were significantly more likely than private patients to be referred to the ED [odds ratio (OR) 1.32, 95% confidence interval (CI) 1.09-1.60]. Private patients who were triaged by a clinician compared to nonclinician staff were over 6 times more likely to be referred to the ED (OR 6.46, 95% CI 4.63-9.01). Medicaid patients were nearly 9 times more likely to have an ED referral when triaged by a clinician (OR 8.72, 95% CI 6.19-12.29). CONCLUSIONS: Only one-third of UCCs across the United States referred an apparent emergent surgical case to the ED, potentially delaying care. Medicaid patients were more likely to be referred directly to the ED versus privately insured patients. All patients triaged by clinicians were significantly more likely to be referred to the ED; however, the disparity between private and Medicaid patients remained.

Diagnostic whole genome sequencing and split-read mapping for nucleotide resolution breakpoint identification in CNTNAP2 deficiency syndrome.

Whole genome sequencing (WGS) has the potential to report on all types of genetic abnormality, thus converging diagnostic testing on a single methodology. Although WGS at sufficient depth for robust detection of point mutations is still some way from being affordable for diagnostic purposes, low-coverage WGS is already an excellent method for detecting copy number variants ("CNVseq"). We report on a family in which individuals presented with a presumed autosomal recessive syndrome of severe intellectual disability and epilepsy. Array comparative genomic hybridization (CGH) analysis had revealed a homozygous deletion apparently lying within intron 3 of CNTNAP2. Since this was too small for confirmation by FISH, CNVseq was used, refining the extent of this mutation to approximately 76.8 kb, encompassing CNTNAP2 exon 3 (an out-of-frame deletion). To characterize the precise breakpoints and provide a rapid molecular diagnostic test, we resequenced the CNVseq library at medium coverage and performed split read mapping. This yielded information for a multiplex polymerase chain reaction (PCR) assay, used for cascade screening and/or prenatal diagnosis in this family. This example demonstrates a rapid, low-cost approach to converting molecular cytogenetic findings into robust PCR-based tests.

Acentric chromosome congression and alignment on the metaphase plate via kinetochore-independent forces in Drosophila.

Chromosome congression and alignment on the metaphase plate involves lateral and microtubule plus-end interactions with the kinetochore. Here we take advantage of our ability to efficiently generate a GFP-marked acentric X chromosome fragment in Drosophila neuroblasts to identify forces acting on chromosome arms that drive congression and alignment. We find acentrics efficiently align on the metaphase plate, often more rapidly than kinetochore-bearing chromosomes. Unlike intact chromosomes, the paired sister acentrics oscillate as they move to and reside on the metaphase plate in a plane distinct and significantly further from the main mass of intact chromosomes. Consequently, at anaphase onset acentrics are oriented either parallel or perpendicular to the spindle. Parallel-oriented sisters separate by sliding while those oriented perpendicularly separate via unzipping. This oscillation, together with the fact that in monopolar spindles acentrics are rapidly shunted away from the poles, indicates that distributed plus-end directed forces are primarily responsible for acentric migration. This conclusion is supported by the observation that reduction of EB1 preferentially disrupts acentric alignment. In addition, reduction of Klp3a activity, a gene required for the establishment of pole-to-pole microtubules, preferentially disrupts acentric alignment. Taken together these studies suggest that plus-end forces mediated by the outer pole-to-pole microtubules are primarily responsible for acentric metaphase alignment. Surprisingly, we find that a small fraction of sister acentrics are anti-parallel aligned indicating that the kinetochore is required to ensure parallel alignment of sister chromatids. Finally, we find induction of acentric chromosome fragments results in a global reorganization of the congressed chromosomes into a torus configuration. Article Summary: The kinetochore serves as a site for attaching microtubules and allows for successful alignment, separation, and segregation of replicated sister chromosomes during cell division. However, previous studies have revealed that sister chromosomes without kinetochores (acentrics) often align to the metaphase plate, undergo separation and segregation, and are properly transmitted to daughter cells. In this study, we discuss the forces acting on chromosomes, independent of the kinetochore, underlying their successful alignment in early mitosis.

Removing Barriers and Honoring Autonomy: Rethinking Mental Health Professional Assessments in Adolescent Gender-Affirming Medical Care.

ABSTRACT: Adolescents seeking gender-affirming medical care (GAMC) face numerous barriers that may delay or inhibit their access to these services. Such obstacles include mental health professional (MHP) assessment requirements prior to initiating GAMC. MHP letters ultimately carry little benefit for patients. Their formulaic nature discourages nuance, reduces likelihood of capturing gender embodiment goals (beyond a narrow definition of gender dysphoria), and may cause clinicians to overlook presenting mental health concerns. MHP assessment requirements also reinforce the conception of gender dysphoria as a mental health disorder. Moreover, studies have not shown that requiring MHP assessment letters effectively reduces regret among patients. Fortunately, primary clinicians who provide GAMC are most often capable of assessing patients without additional input from an MHP. In this article, we provide an ethical framework for clinicians that prioritizes patient autonomy through an informed assent approach. We discuss Appelbaum's criteria and its application, and contexts in which MHP consultation is appropriate. We also address common questions about informed assent among clinicians, patients, and families. Finally, we advocate for bolstering multidisciplinary support teams involved in GAMC to facilitate the informed assent process. This approach upholds patient autonomy, expands access to GAMC, and utilizes the mental health workforce more effectively.

A Patient-Centered Documentation Skills Curriculum for Preclerkship Medical Students in an Open Notes Era.

Introduction: New legislation allows patients (with permitted exceptions) to read their clinical notes, leading to both benefits and ethical dilemmas. Medical students need a robust curriculum to learn documentation skills within this challenging context. We aimed to teach note-writing skills through a patient-centered lens with special consideration for the impact on patients and providers. We developed this session for first-year medical students within their foundational clinical skills course to place bias-free language at the forefront of how they learn to construct a medical note. Methods: One hundred seventy-three first-year medical and dental students participated in this curriculum. They completed an asynchronous presession module first, followed by a 2-hour synchronous workshop including a didactic, student-led discussion and sample patient note exercise. Students were subsequently responsible throughout the year for constructing patient-centered notes, graded by faculty with a newly developed rubric and checklist of best practices. Results: On postworkshop surveys, learners reported increased preparedness in their ability to document in a patient-centered manner (presession M = 2.2, midyear M = 3.9, p < .001), as rated on a 5-point Likert scale (1 = not prepared at all, 5 = very prepared), and also found this topic valuable to learn early in their training. Discussion: This curriculum utilizes a multipart approach to prepare learners to employ clinical notes to communicate with patients and providers, with special attention to how patients and their care partners receive a note. Future directions include expanding the curriculum to higher levels of learning and validating the developed materials.

Secret shopper studies: an unorthodox design that measures inequities in healthcare access.

Secret shopper studies are particularly potent study designs that allow for the gathering of objective data for a variety of research hypotheses, including but not limited to, healthcare delivery, equity of healthcare, and potential barriers to care. Of particular interest during the COVID-19 pandemic, secret shopper study designs allow for the gathering of data over the phone. However, there is a dearth of literature available on appropriate methodological practices for these types of studies. To make these study designs more widely accessible, here we outline the case for using the secret shopper methodology and detail best practices for designing and implementing them.

Charges for Initial Visits for Uninsured Patients at Musculoskeletal Urgent Care Centers in the US.

Importance: In recent years, specialized musculoskeletal urgent care centers (MUCCs) have opened across the US. Uninsured patients may increasingly turn to these orthopedic-specific urgent care centers as a lower-cost alternative to emergency department or general urgent care center visits. Objective: To assess out-of-pocket costs and factors associated with these costs at MUCCs for uninsured and underinsured patients in the US. Design, Setting, and Participants: In this survey study, a national secret shopper survey was conducted in June 2019. Clinics identified as MUCCs in 50 states were contacted by telephone by investigators using a standardized script and posing as uninsured patients seeking information on the out-of-pocket charge for a new patient visit. Exposures: State Medicaid expansion status, clinic Medicaid acceptance status, state Medicaid reimbursement rate, median income per zip code, and clinic region. Main Outcomes and Measures: The primary outcome was each clinic's out-of-pocket charge for a level 3 visit, defined as a new patient office visit requiring medical decision-making of low complexity. Linear regression was used to examine correlations of price with clinic policy against accepting Medicaid, median income per zip code, and Medicaid reimbursement for a level 3 visit. Results: Of 565 MUCCs identified, 558 MUCCs were able to be contacted (98.8%); 536 of the 558 MUCCs (96.1%) disclosed a new patient visit out-of-pocket charge. Of those, 313 (58.4%) accepted Medicaid insurance and 326 (60.8%) were located in states with expanded Medicaid at the time of the survey. The mean (SD) price of a visit to an MUCC was $250 ($110). Clinic policy against accepting Medicaid (ß, 22.91; 95% CI, 12.57-33.25; P < .001), higher median income per zip code (ß, 0.00056; 95% CI, 0.00020-0.00092; P = .003), and increased Medicaid reimbursement for a level 3 visit (ß, 0.737; 95% CI, 0.158-1.316; P = .01) were positively correlated with visit price. The overall regression was statistically significance (R2 = 0.084; P < .001). Conclusions and Relevance: In this survey study, MUCCs charged a mean price of $250 for a new patient visit. Medicaid acceptance policy, median income per zip code, and Medicaid reimbursement for a level 3 visit were associated with differences in out-of-pocket charges. These findings suggest that accessibility to orthopedic urgent care at MUCCs may be limited for underinsured and uninsured patients.

Validity of administrative database coding for kidney disease: a systematic review.

BACKGROUND: Information in health administrative databases increasingly guides renal care and policy. STUDY DESIGN: Systematic review of observational studies. SETTING & POPULATION: Studies describing the validity of codes for acute kidney injury (AKI) and chronic kidney disease (CKD) in administrative databases operating in any jurisdiction. SELECTION CRITERIA: After searching 13 medical databases, we included observational studies published from database inception though June 2009 that validated renal diagnostic and procedural codes for AKI or CKD against a reference standard. INDEX TESTS: Renal diagnostic or procedural administrative data codes. REFERENCE TESTS: Patient chart review, laboratory values, or data from a high-quality patient registry. RESULTS: 25 studies of 13 databases in 4 countries were included. Validation of diagnostic and procedural codes for AKI was present in 9 studies, and validation for CKD was present in 19 studies. Sensitivity varied across studies and generally was poor (AKI median, 29%; range, 15%-81%; CKD median, 41%; range, 3%-88%). Positive predictive values often were reasonable, but results also were variable (AKI median, 67%; range, 15%-96%; CKD median, 78%; range, 29%-100%). Defining AKI and CKD by only the use of dialysis generally resulted in better code validity. The study characteristic associated with sensitivity in multivariable meta-regression was whether the reference standard used laboratory values (P < 0.001); sensitivity was 39% lower when laboratory values were used (95% CI, 23%-56%). LIMITATIONS: Missing data in primary studies limited some of the analyses that could be done. CONCLUSIONS: Administrative database analyses have utility, but must be conducted and interpreted judiciously to avoid bias arising from poor code validity.

A Course-Based Undergraduate Research Experience in CRISPR-Cas9 Experimental Design to Support Reverse Genetic Studies in Arabidopsis thaliana.

Gene-editing tools such as CRISPR-Cas9 have created unprecedented opportunities for genetic studies in plants and animals. We designed a course-based undergraduate research experience (CURE) to train introductory biology students in the concepts and implementation of gene-editing technology as well as develop their soft skills in data management and scientific communication. We present two versions of the course that can be implemented with twice-weekly meetings over a 5-week period. In the remote-learning version, students performed homology searches, designed guide RNAs (gRNAs) and primers, and learned the principles of molecular cloning. This version is appropriate when access to laboratory equipment or in-person instruction is limited, such as during closures that have occurred in response to the COVID-19 pandemic. In person, students designed gRNAs, cloned CRISPR-Cas9 constructs, and performed genetic transformation of Arabidopsis thaliana. Students learned how to design effective gRNA pairs targeting their assigned gene with an 86% success rate. Final exams tested students' ability to apply knowledge of an unfamiliar genome database to characterize gene structure and to properly design gRNAs. Average final exam scores of ∼73% and ∼84% for in-person and remote-learning CUREs, respectively, indicated that students met learning outcomes. The highly parallel nature of the CURE makes it possible to target dozens to hundreds of genes, depending on the number of sections. Applying this approach in a sensitized mutant background enables focused reverse genetic screens for genetic suppressors or enhancers. The course can be adapted readily to other organisms or projects that employ gene editing.

In vitro antimicrobial activity of silver-processed catheters for neurosurgery.

OBJECTIVES: To investigate the in vitro antibacterial activity of silver-processed catheters for use in neurosurgery using clinically predictive tests. METHODS: The antimicrobial activity of a commercially available silver-processed external ventricular drain catheter was evaluated against Staphylococcus epidermidis, methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli and Propionibacterium acnes. Non-impregnated catheters were used as controls. Two assays were performed: (i) testing the ability of the catheter to kill 100% of the attached bacteria (tK100); and (ii) in vitro challenge to determine the ability to prevent colonization under flow conditions. High and low inocula (10(4) and 10(7) cfu/mL) were used. Silver-processed and control catheters were examined by scanning electron microscopy and focused ion beam scanning electron microscopy; electron back-scatter and energy-dispersive X-ray analyses were used to investigate the distribution of silver within the processed catheter. RESULTS: The silver-processed catheters were not able to kill any of the bacteria tested in the tK100 assay at high inoculum. At low inoculum S. epidermidis was eradicated and some activity was seen against E. coli but without complete eradication. MRSA was also not eradicated even at low inoculum. The in vitro challenge test showed no prevention of colonization for any of the strains. Silver particles were shown to be >500 nm in size. CONCLUSIONS: The commercial silver-impregnated catheter was not able to eradicate MRSA or E. coli and while it showed activity against S. epidermidis in one assay it was unable to prevent colonization in vitro under in-flow conditions. This is consistent with clinical studies on silver-processed catheters.

COVID-19 testing capabilities at urgent care centers in states with greatest disease burden.

While rapid and accessible diagnosis is paramount to monitoring and reducing the spread of disease, COVID-19 testing capabilities across the U.S. remain constrained. For many individuals, urgent care centers (UCCs) may offer the most accessible avenue to be tested. Through a phone survey, we describe the COVID-19 testing capabilities at UCCs and provide a snapshot highlighting the limited COVID-19 testing capabilities at UCCs in states with the greatest disease burden.

The dark side of competition: How competitive behaviour and striving to avoid inferiority are linked to depression, anxiety, stress and self-harm.

This study was guided by the social rank theory of depression and aimed to explore the relationship between depression, anxiety, stress and self-harm with striving to avoid inferiority, feelings of shame and styles of attachment. Participants diagnosed with depression (n = 62) completed a series of questionnaires measuring striving to avoid inferiority, fears of missing out, being overlooked and active rejection, attachment, social rank and psychopathologies. Striving to avoid inferiority was significantly linked to social rank variables and anxious attachment. Mediator analyses revealed that the relationship between striving to avoid inferiority and depression was mediated by the social rank variable of external shame, and also anxious attachment. These findings suggest that elevated competitive behaviour can have a 'dark side'. When people feel insecure in their social environments, it can focus them on a hierarchical view of themselves and others, with a fear of rejection if they feel they have become too inferior or subordinate. This may increase vulnerability to depression, anxiety and stress.

Development of a striving to avoid inferiority scale.

Social rank theory suggests that mood variation is linked to the security a person feels in his/her social domain and the extent to which they are sensitive to involuntary subordination (e.g. feeling defeated and feeling inferior). Previous studies looking at rank-related and competitive behaviour have often focused on striving for dominance, whereas social rank theory has focused on striving to avoid inferiority. This study set out to develop a measure of 'Striving to Avoid Inferiority' (SAIS) and assess its relationship to other rank and mood-related variables. We hypothesized two factors: one we called insecure striving, relating to fear of rejection/criticism for 'not keeping up', and the second we called secure non-striving, relating to feeling socially acceptable and valued regardless of whether one succeeds or not. This scale was given to 207 undergraduates. The SAIS had good psychometric properties, with the two factors of insecure striving and secure non-striving strongly supported by exploratory factor analysis. Both factors were significantly (though contrastingly) related to various fears of rejection, need for validation, hypercompetitive attitudes, feeling inferior to others, submissive behaviour and indicators of stress, anxiety and depression. Striving to avoid inferiority was a significant predictor of psychopathologies, especially where individuals perceived themselves to have low social rank.

A Large Intragenic Deletion in the ACADM Gene Can Cause MCAD Deficiency but is not Detected on Routine Sequencing.

We report of a family who has three members affected by medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, one of whom sadly died in the neonatal period prior to diagnosis. Routine sequencing, available on a service basis in the UK, identified only a heterozygous mutation in ACADM gene (c.985A>G, p.Lys329Glu) in this family. Linkage analysis suggested a possible intragenic deletion which was confirmed by the use of array-based comparative genomic hybridization (aCGH). This second mutation was a large intragenic deletion encompassing at least exons 1-6 of the ACADM gene. Now that this deletion has been identified, several family members have come forward for carrier testing which was not possible previously. Larger deletions (20bp or more) have only previously been reported twice, but these may be a more frequent cause of MCAD deficiency than hitherto believed, due to fact that these are not anticipated and, therefore, the routine diagnostic techniques used will not identify them. This finding represents a useful learning point in the management of families with MCAD deficiency, and highlights that we should be routinely looking for larger deletions, when only one of the mutations can be identified on standard sequencing.