Characterization of a novel deep-intronic variant in DYNC2H1 identified by whole-exome sequencing in a patient with a lethal form of a short-rib thoracic dysplasia type III.

Biallelic pathogenic variants in DYNC2H1 are the cause of short-rib thoracic dysplasia type III with or without polydactyly (OMIM #613091), a skeletal ciliopathy characterized by thoracic hypoplasia due to short ribs. In this report, we review the case of a patient who was admitted to the Neonatal Intensive Care Unit (NICU) of Indiana University Health (IUH) for respiratory support after experiencing respiratory distress secondary to a small, narrow chest causing restrictive lung disease. Additional phenotypic features include postaxial polydactyly, short proximal long bones, and ambiguous genitalia were noted. Exome sequencing (ES) revealed a maternally inherited likely pathogenic variant c.10322C > T p.(Leu3448Pro) in the DYNC2H1 gene. However, there was no variant found on the paternal allele. Microarray analysis to detect deletion or duplication in DYNC2H1 was normal. Therefore, there was insufficient evidence to establish a molecular diagnosis. To further explore the data and perform additional investigations, the patient was subsequently enrolled in the Undiagnosed Rare Disease Clinic (URDC) at Indiana University School of Medicine (IUSM). The investigators at the URDC performed a reanalysis of the ES raw data, which revealed a paternally inherited DYNC2H1 deep-intronic variant c.10606-14A > G predicted to create a strong cryptic acceptor splice site. Additionally, the RNA sequencing of fibroblasts demonstrated partial intron retention predicted to cause a premature stop codon and nonsense-mediated mRNA decay (NMD). Droplet digital RT-PCR (RT-ddPCR) showed a drastic reduction by 74% of DYNCH2H1 mRNA levels. As a result, the intronic variant was subsequently reclassified as likely pathogenic resulting in a definitive clinical and genetic diagnosis for this patient. Reanalysis of ES and fibroblast mRNA experiments confirmed the pathogenicity of the splicing variants to supplement critical information not revealed in original ES or CMA reports. The NICU and URDC collaboration ended the diagnostic odyssey for this family; furthermore, its importance is emphasized by the possibility of prenatally diagnosing the mother's current pregnancy.

Use of the PET ligand florbetapir for in vivo imaging of pancreatic islet amyloid deposits in hIAPP transgenic mice.

AIMS/HYPOTHESIS: Islet amyloid deposits contribute to beta cell dysfunction and death in most individuals with type 2 diabetes but non-invasive methods to determine the presence of these pathological protein aggregates are currently not available. Therefore, we examined whether florbetapir, a radiopharmaceutical agent used for detection of amyloid-ß deposits in the brain, also allows identification of islet amyloid in the pancreas. METHODS: Saturation binding assays were used to determine the affinity of florbetapir for human islet amyloid polypeptide (hIAPP) aggregates in vitro. Islet amyloid-prone transgenic mice that express hIAPP in their beta cells and amyloid-free non-transgenic control mice were used to examine the ability of florbetapir to detect islet amyloid deposits in vitro, in vivo and ex vivo. Mice or mouse pancreases were subjected to autoradiographic, histochemical and/or positron emission tomography (PET) analyses to assess the utility of florbetapir in identifying islet amyloid. RESULTS: In vitro, florbetapir bound synthetic hIAPP fibrils with a dissociation constant of 7.9 nmol/l. Additionally, florbetapir bound preferentially to amyloid-containing hIAPP transgenic vs amyloid-free non-transgenic mouse pancreas sections in vitro, as determined by autoradiography (16,475 ± 5581 vs 5762 ± 575 density/unit area, p < 0.05). In hIAPP transgenic and non-transgenic mice fed a high-fat diet for 1 year, intravenous administration of florbetapir followed by PET scanning showed that the florbetapir signal was significantly higher in amyloid-laden hIAPP transgenic vs amyloid-free non-transgenic pancreases in vivo during the first 5 min of the scan (36.83 ± 2.22 vs 29.34 ± 2.03 standardised uptake value × min, p < 0.05). Following PET, pancreases were excised and florbetapir uptake was determined ex vivo by γ counting. Pancreatic uptake of florbetapir was significantly correlated with the degree of islet amyloid deposition, the latter assessed by histochemistry (r = 0.74, p < 0.001). CONCLUSIONS/INTERPRETATION: Florbetapir binds to islet amyloid deposits in a specific and quantitative manner. In the future, florbetapir may be useful as a non-invasive tool to identify islet amyloid deposits in humans.

MLH1-rheMac hereditary nonpolyposis colorectal cancer syndrome in rhesus macaques.

Over the past two decades, 33 cases of colonic adenocarcinomas have been diagnosed in rhesus macaques (Macaca mulatta) at the nonhuman primate colony of the Keeling Center for Comparative Medicine and Research at The University of Texas MD Anderson Cancer Center. The distinctive feature in these cases, based on PET/computed tomography (CT) imaging, was the presence of two or three tumor lesions in different locations, including proximal to the ileocecal juncture, proximal to the hepatic flexure, and/or in the sigmoid colon. These colon carcinoma lesions selectively accumulated [18F]fluorodeoxyglucose ([18F]FDG) and [18F]fluoroacetate ([18F]FACE) at high levels, reflecting elevated carbohydrate and fatty acid metabolism in these tumors. In contrast, the accumulation of [18F]fluorothymidine ([18F]FLT) was less significant, reflecting slow proliferative activity in these tumors. The diagnoses of colon carcinomas were confirmed by endoscopy. The expression of MLH1, MSH2, and MSH6 proteins and the degree of microsatellite instability (MSI) was assessed in colon carcinomas. The loss of MLH1 protein expression was observed in all tumors and was associated with a deletion mutation in the MLH1 promoter region and/or multiple single-nucleotide polymorphism (SNP) mutations in the MLH1 gene. All tumors exhibited various degrees of MSI. The pedigree analysis of this rhesus macaque population revealed several clusters of affected animals related to each other over several generations, suggesting an autosomal dominant transmission of susceptibility for colon cancer. The newly discovered hereditary nonpolyposis colorectal cancer syndrome in rhesus macaques, termed MLH1-rheMac, may serve as a model for development of novel approaches to diagnosis and therapy of Lynch syndrome in humans.

Kirschner Wire Breakage during Removal Requiring Retrieval.

Kirschner wires (K-wires) are widely used for fixation of fractures and dislocations in the hand as they are readily available, reliable, and cost-effective. Complication rates of up to 18% have been reported. However, K-wire breakage during removal is rare. We present one such case illustrating a simple technique for retrieval. A 35-year-old male presented with a distal phalanx fracture of his right middle finger. This open fracture was treated with K-wire fixation. Postoperatively, he developed a pin site infection with associated finger swelling. The K-wire broke during removal with the proximal piece completely retained in his middle phalanx. To minimise risk of osteomyelitis, the K-wire was removed with a novel surgical technique. He had full return of hand function. Intraoperative K-wire breakage has a reported rate of 0.1%. In our case, there was no obvious cause of breakage and the patient denied postoperative trauma. On the other hand, pin site infections are much more common with reported rates of up to 7% in the hand or wrist. K-wire fixation is a simple method for bony stabilisation but can be a demanding procedure with complications often overlooked. It is important to be aware of the potential sequelae.

Reconstruction of complex orbital exenteration defects: a single center's experience with a five-year follow-up.

BACKGROUND: Orbital exenteration presents a challenge to the reconstructive surgeon. Defects may be treated with split skin grafts, local advancement flaps, or free flap coverage. There are few published series showing the long-term outcomes of reconstruction. Our results, with at least a 5-year follow-up, are presented. METHODS: A retrospective review of 15 immediate reconstructions after orbital exenterations for malignancies in 12 patients at a tertiary referral center over a 5-year period, was done. All flaps were followed up for at least 5 years (mean, 75 months; range, 3-118 months). RESULTS: Malignancies included squamous cell carcinoma, basal cell carcinoma, meningioma, sebaceous gland carcinoma, and rhabdomyosarcoma. Eight cervicofacial rotation-advancement flaps and 4 anterolateral thigh, 1 rectus abdominis, and 1 radial forearm free tissue transfers were used. Aggressive postoperative radiotherapy (9/15) was well tolerated by both regional and free flaps. Both cervicofacial flaps in previously irradiated patients had wound dehiscence or fistula formation. Six (50%) patients died during follow-up, 4 of whom (33%) died of tumor recurrence. CONCLUSIONS: Flap reconstruction after complex orbital exenteration is associated with low morbidity. Cervicofacial rotation-advancement flaps offer reliable, single-stage, aesthetically pleasing reconstructions. They should be avoided in the previously irradiated. Free tissue transfer is indicated for volume replacement, after previous radiotherapy, after tumor recurrence and previous use of locoregional flaps. Reconstruction of complex orbital exenteration defects for malignancies should be undertaken in centers with experience in the management of these procedures.

Skin graft meshing, over-meshing and cross-meshing.

INTRODUCTION: Split skin grafts (SSGs) are often meshed to increase their size and allow exudate to escape. We investigated the expansion obtained with meshing, and the possibility of re-meshing skin that has already been meshed ("overmeshing"). Both useful and inadvisable permutations are illustrated. MATERIAL AND METHODS: Thin porcine SSGs were sideways meshed, or meshed with ratios of 1.5:1 and 3:1. Subsequently samples were over-meshed in a variety of ratios and directions. All grafts were maximally expanded and their areas calculated. RESULTS: Meshed skin did not expand as much as suggested by the ratios displayed on dermacarriers. A 1:1.5 dermacarrier produced an area expansion of 1.36×, and a 1:3 meshing apparatus produced only a 1.80×area expansion. Several combinations of twice-meshed SSGs maintained integrity as long as over-meshing was done in the axis of initial meshing. Up to 2.3×expansion was obtained, by following a 1:1.5 mesh with a 1:3 mesh. We term this procedure as "overmeshing". Re-meshing in a direction orthogonal to initial meshing (cross meshing) cut the skin into small pieces. CONCLUSION: Over-meshing a SSG can allow considerable further expansion, facilitating overgrafting of donor sites or simply increasing the area that can be covered with the existing harvested skin.

Epitendinous suture techniques in extensor tendon repairs--an experimental evaluation.

PURPOSE: The tension-band principle might be relevant to extensor tendon repairs, and a dorsal-only Silfverskiöld epitendinous repair is stronger and stiffer than more conventional techniques in vitro. We aimed to evaluate the strength and stiffness of the strongest epitendinous sutures described, using an in vitro model that subjects the repair to angular force over a pulley, thereby creating a tension-band model. METHODS: Silfverskiöld dorsal-only epitendinous extensor tendon repairs in porcine foot tendons (n = 8) were compared to reverse (buried) Silfverskiöld (n = 8), Halsted (n = 8), and interrupted horizontal mattress (IHM) repairs (n = 6) in vitro with a tensiometer around a 45° pulley. Thirty tendons total were tested to assess the force required for 2-mm gapping and ultimate tensile strength. RESULTS: The IHM repair had a significantly higher ultimate tensile strength (43 N; SD, 10 N) than the other repairs, which had strengths between 27 N (SD, 4 N) and 31 N (SD, 7 N). The IHM was also significantly more resistant to gapping than the Silfverskiöld and Halsted repairs. CONCLUSIONS: Interlocking horizontal mattress, dorsal-only extensor tendon repairs were significantly stronger and more resistant to gapping than Silfverskiöld and Halsted repairs. Other repairs were still strong and resistant to gapping in comparison to previously published data for conventional repairs. CLINICAL RELEVANCE: The IHM is a relatively difficult technique to perform, and it remains to be seen whether the additional strength translates to clinical benefits over the easier Silfverskiöld technique.

Inflammatory posterior interosseous nerve palsy in a patient with psoriatic arthropathy.

Psoriasis is a chronic, relapsing, inflammatory skin disorder with a strong genetic basis. Five patterns of psoriatic arthritis have been identified: asymmetrical oligoarticular arthritis, symmetrical polyarthritis, distal interphalangeal arthropathy, arthritis mutilans and spondylitis with or without sacroiliitis. Extra-articular disease is uncommon. We report a rare case of an inflammatory posterior interosseus nerve palsy in a patient with known psoriatic arthropathy, where investigation warranted medical treatment over a surgical approach. The commonest cause of posterior interosseus nerve palsy is entrapment at the proximal forearm. Other possible aetiologies include extension of elbow synovitis as described in rheumatoid arthritis, trauma eg. Monteggia fractures, tumours and iatrogenic injuries. We discuss the diagnostic dilemma and the management issues for upper limb surgeons.

Scarring occurs at a critical depth of skin injury: precise measurement in a graduated dermal scratch in human volunteers.

BACKGROUND: The association between scarring and the depth of dermal injury or burn is clinically recognized but not quantified. The authors tested the hypothesis that there is a critical depth beyond which a fibrous scar develops. METHODS: A novel jig produced a wound that was deep dermal at one end and superficial dermal at the other. Pilot studies in cadaveric and ex vivo breast skin confirmed the depth of injury. Healthy volunteers had a standardized dermal wound made on the lateral aspect of the hip. Digital photography recorded the surface appearance of wound healing and scar development. High-frequency ultrasound demonstrated the depth of the healing wound and subsequent scar in vivo. RESULTS: One hundred thirteen human subjects participated in the clinical study. Mean length of follow up was 28.6 +/- 13.2 weeks. The deep dermal end of the wound healed with a visible scar and the superficial end had no visible residual mark after week 18. The initial length of injury was 51.3 +/- 0.6 mm, which reduced to a scar of 34.9 +/- 1.0 mm at 36 weeks (corresponding areas were 196.6 +/- 7.5 mm and 92.7 +/- 9.4 mm). High-frequency ultrasound analysis showed a gradual reduction in scar thickness at the deep end and no detectable scar at the shallow end. The transition point between scar and no scar marked the threshold depth for scarring. This was calculated as 0.56 +/- 0.03 mm, or 33.1 percent of normal hip skin thickness. CONCLUSIONS: The dermal scratch provides a well-tolerated, standardized, and reproducible wound model for investigating the healing response to dermal injury of different depths. There is a threshold depth of dermal injury at which scarring develops.

Pituitary hypoplasia and respiratory distress syndrome in Prop1 knockout mice.

Mutations in Prophet of PIT1 (Prop1), one of several homeodomain transcription factors that are required for the development of the anterior pituitary gland, are the predominant cause of MPHD (multiple pituitary hormone deficiency) in humans. We show that deletion of Prop1 in mice causes severe pituitary hypoplasia with failure of the entire Pit1 lineage and delayed gonadotrope development. The pituitary hormone deficiencies cause secondary endocrine problems and a high rate of perinatal mortality due to respiratory distress. Lung atelectasis in mutants correlates with reduced levels of NKX2.1 and surfactant. Lethality of mice homozygous for either the null allele or a spontaneous hypomorphic allele is strongly influenced by genetic background. Prop1-null mice are an excellent model for MPHD and may be useful for testing the efficacy of pharmaceutical intervention for neonatal respiratory distress.

Topical radiant heating in wound healing: an experimental study in a donor site wound model*.

The importance of temperature in the wound-healing process is rapidly being recognised as a novel way in which to manipulate the wound-healing environment. In this study, we aimed to investigate the direct effect of topical radiant heating (TRH), using a novel bandaging system (Warm-Up, Arizant Health care Inc., Eden Prairie MN, USA; Augustine Medical, USA), on wound healing at a physiological and cellular level. Experimental bandages were positioned over split-thickness skin graft donor site wounds of 12 patients undergoing graft harvesting from the anterior thigh. The experimental group (n=6) underwent intermittent heating for 5 hours (three 1-hour heating cycles at 38 degrees C, separated by two 1-hour rest periods), whilst the control group (n=6) received no radiant heating. Physiological blood-flow recordings both in the control group and the topical radiant heat cohort were undertaken using Laser Doppler Imaging (LDI). Skin biopsies were obtained at identical time points, and immunohistochemical analysis was undertaken using antibodies against neutrophils (NP57), lymphocytes (CD3) and macrophages (CD68). We found that TRH significantly increased local dermal blood flow (P<0.001) by up to 100% in both injured and intact skin. Furthermore, this increase in flow was associated with a significant (P<0.05) increase in CD3 immunoreactivity on day 1 postoperatively. This study demonstrates that TRH increases local blood flow and lymphocyte (CD3) extravasation, and we postulate that these changes may enhance local innate immunity within the healing wound environment.