CRISPR-Cas9/long-read sequencing approach to identify cryptic mutations in BRCA1 and other tumour suppressor genes.

Current clinical approaches for mutation discovery are based on short sequence reads (100-300 bp) of exons and flanking splice sites targeted by multigene panels or whole exomes. Short-read sequencing is highly accurate for detection of single nucleotide variants, small indels and simple copy number differences but is of limited use for identifying complex insertions and deletions and other structural rearrangements. We used CRISPR-Cas9 to excise complete BRCA1 and BRCA2 genomic regions from lymphoblast cells of patients with breast cancer, then sequenced these regions with long reads (>10 000 bp) to fully characterise all non-coding regions for structural variation. In a family severely affected with early-onset bilateral breast cancer and with negative (normal) results by gene panel and exome sequencing, we identified an intronic SINE-VNTR-Alu retrotransposon insertion that led to the creation of a pseudoexon in the BRCA1 message and introduced a premature truncation. This combination of CRISPR-Cas9 excision and long-read sequencing reveals a class of complex, damaging and otherwise cryptic mutations that may be particularly frequent in tumour suppressor genes replete with intronic repeats.

Impact of Donor Hepatitis C Virus on Kidney Transplant Outcomes for Hepatitis C-positive Recipients in the Direct-acting Antiviral Era: Time to Revise the Kidney Donor Risk Index?

BACKGROUND: Kidneys from donors with hepatitis C virus (HCV) infection are traditionally considered to be at risk for poorer survival outcomes, as reflected in the kidney donor profile index (KDPI). Modern direct-acting antivirals may modify this risk. METHODS: Using United Network for Organ Sharing data, HCV-infected adult first-time kidney transplant recipients from 2014 to 2017 were examined. Graft and patient survival were compared in a propensity-matched cohort of recipients of HCV antibody (Ab)(+) kidneys versus Ab(-) kidneys. Subsequent analysis was performed in a propensity-matched cohort of recipients of HCV-viremic (RNA positive) versus HCV-naïve kidneys. RESULTS: There were 379 recipients each in the matched cohort of recipients of HCV Ab(+) versus HCV Ab(-) kidneys. Despite a higher KDPI (58.2% for HCV Ab[+] versus 38.8% for HCV Ab[-]), 1-year patient and graft survival were similar in the HCV(+) and HCV(-) groups (95.4% and 94.9% versus 97.9% and 96.0%, P = 0.543 and P = 0.834, respectively). There were 200 recipients each in the cohort of recipients of HCV-viremic versus HCV-naïve kidneys, with the KDPI again higher in the HCV-viremic group (56.8% versus 35.2%). Baseline hazard ratios (HRs) for graft failure (HR, 4.69; P = 0.009) and death (HR, 7.60; P = 0.003) were significantly elevated in the viremic group, but crossed 1 at 21 and 24 months, respectively. CONCLUSIONS: In the modern direct-acting antiviral era, calculated likely KDPI overestimates risk kidneys from HCV (+) donors. Donor viremia conveys an early risk which appears to subside over time. These results suggest that it may be time to revise the kidney donor risk index.

The role of FDG-PET in detecting rejection after liver transplantation.

BACKGROUND: The activation and increased metabolic activity of T cells in acute cellular rejection could allow fluoro-2-deoxyglucose positron emission tomography to be utilized for detection of acute cellular rejection. The objective of this study was to evaluate the effectiveness of fluoro-2-deoxyglucose positron emission tomography in detecting acute cellular rejection in the clinical setting. METHODS: Fluoro-2-deoxyglucose positron emission tomography studies were performed on 88 orthotopic liver transplant patients at 7 and 17 days postoperatively (first positron emission tomography and second positron emission tomography, respectively). Additional studies were performed if patients had suspicion of rejection and at resolution of rejection (third positron emission tomography and fourth positron emission tomography, respectively). A circular region of interest was placed over the liver for semiquantitative evaluation of fluoro-2-deoxyglucose positron emission tomography images by means of standard uptake values. RESULTS: Eighteen of 88 patients in our study (20.5%) had histologically proven acute cellular rejection during a 16 ± 11 day follow-up. There was no significant difference between the standard uptake values of first positron emission tomography among non-rejecters versus rejecters (2.05 ±0.46 non-rejecters versus 1.82 ± 0.40 rejecters, P = .127). Within the rejection cohort, the standard uptake values from the third positron emission tomography (rejection) were higher compared to the first positron emission tomography (baseline) (2.41 ± 0.48 third positron emission tomography versus 1.82 ± 0.41 first positron emission tomography, P < .001). CONCLUSION: Increased signal on fluoro-2-deoxyglucose positron emission tomography over baseline is associated with acute cellular rejection in liver transplant recipients. Additional prospective validation studies are essential to define the role of fluoro-2-deoxyglucose positron emission tomography scan as an early marker for acute cellular rejection.

Hand-assisted laparoscopic nephrectomy for polycystic kidney disease.

BACKGROUND AND OBJECTIVES: Historically, nephrectomy for autosomal dominant polycystic kidney disease was performed by an open technique. We performed this study to compare outcomes in hand-assisted laparoscopic nephrectomy with open nephrectomy in this population. METHODS: Charts of patients with autosomal dominant polycystic kidney disease who underwent nephrectomy by a transplant surgeon from January 1, 2000, to December 31, 2011, were reviewed. The hand-assisted laparoscopic nephrectomy group was compared with the open group. Data collected included unilateral versus bilateral nephrectomy, operative time, complications, transfusion requirement, and length of stay. RESULTS: Of the 78 patients identified, 18 underwent open transabdominal nephrectomy, 56 underwent hand-assisted laparoscopic nephrectomy, and 2 underwent hand-assisted laparoscopic nephrectomy that was converted to an open procedure. Two patients were excluded because another major procedure was performed at the same time as the nephrectomy. Operative times were similar. Patients undergoing open bilateral nephrectomy were more likely to receive transfusion (odds ratio, 3.57 [95% confidence interval, 0.74-17.19]; P = .016), and the length of stay was longer in the open groups (5.9 days vs 4.0 days for unilateral [P = .013] and 7.8 days vs 4.6 days for bilateral [P = .001]). Overall complication rates were similar. The most frequent complications associated with hand-assisted laparoscopic nephrectomy were the development of an incisional hernia at the hand-port site and arteriovenous fistula thrombosis. CONCLUSION: Hand-assisted laparoscopic nephrectomy can be safely performed without increased operative times or complications. The hand-assisted laparoscopic nephrectomy group enjoyed a shorter length of stay, and fewer patients in this group received transfusion. For patients considering renal transplantation, avoidance of transfusion is important to prevent sensitization and limiting access to compatible organs.

The impact of recipient obesity on outcomes after renal transplantation.

BACKGROUND: The benefit of renal transplantation in obese patients is controversial, with many centers setting upper limits on body mass index (BMI) in consideration for listing patients for transplant. This study was undertaken to determine the effect of recipient obesity on delayed graft function (DGF) and graft survival after renal transplantation. METHODS: Retrospective review of all renal transplant recipients in the United Network for Organ Sharing database from January 1, 2004, through December 31, 2009, was performed. Primary endpoints were DGF and non-death-censored graft survival. Comparisons were made on the basis of the following weight classes: nonobese (BMI < 30), class I obese (30 ≤ BMI < 35), class II obese (35 ≤ BMI < 40), and class III obese (BMI ≥ 40). RESULTS: Multivariable logistic regression indicated a significantly increased risk for DGF in obese patients. The odds ratios for DGF compared with nonobese patients were 1.34 [95% confidence interval (CI) 1.27-1.42; P < 0.001], 1.68 (95% CI 1.56-1.82; P < 0.001), and 2.68 (95% CI 2.34-3.07; P < 0.001) for the class I obese, class II obese, and class III obese groups, respectively. Class I obesity was not a significant risk for non-death-censored graft failure [hazard ratio (HR) 1.00, 95% CI 0.95-1.05; P = 0.901] compared with nonobese patients. Patients in the class II obese (HR 1.15, 95% CI 1.07-1.24; P < 0.001) and class III obese (HR 1.26, 95% CI 1.11-1.43; P < 0.001) groups were at a significantly increased risk for graft failure than their nonobese counterparts. CONCLUSIONS: Obese patients in all weight classes are at an increased risk for DGF after renal transplantation, although differences in non-death-censored graft survival are such that transplantation should not be denied on the basis of BMI criteria alone.

A review of the United States experience with combined heart-liver transplantation.

Since first described by Starzl, combined heart and liver transplantation (CHLT) has been a relatively rare event, although utilization has increased in the past decade. This study was undertaken to review the United States experience with this procedure; UNOS data on CHLT was reviewed. CHLT was compared with liver transplantation alone and heart transplantation alone in terms of acute rejection within 12 months, graft survival, and patient survival. Survival was calculated according to Kaplan-Meier and Cox proportional hazards. Continuous variables were compared using Student's t-test and categorical variables with chi-squared. Between 1987 and 2010, there were 97 reported cases of CHLT in the United States. Amyloidosis was the most common indication for both heart (n = 26, 26.8%) and liver (n = 27, 27.8%) transplantation in this cohort. Liver graft survival in the CHLT cohort at 1, 5, and 10 years was 83.4%, 72.8%, and 71.0%, whereas survival of the cardiac allograft was 83.5%, 73.2%, and 71.5%. This was similar to graft survival in liver alone transplantation (79.4%, 71.0%, 65.1%; P = 0.894) and heart transplantation alone (82.6%, 71.9%, 63.2%; P = 0.341). CHLT is a safe and effective procedure, with graft survival rates similar to isolated heart and isolated liver transplantation.

Employment outcomes following successful renal transplantation.

BACKGROUND: Data on employment outcomes after successful renal transplantation are few. We conducted this study to identify favorable factors for employment after transplantation. METHODS: Adult patients <65 yr of age who underwent renal transplantation between January 1, 2002 and December 31, 2007 were surveyed. Patients with graft survival <1 yr were excluded. We also tested their knowledge of Medicare coverage after transplantation. Data were analyzed using chi-squared and Fisher's exact tests. p-Value <0.05 was considered statistically significant. RESULTS: A 55% response rate was obtained where 56% of respondents were employed after transplantation. Race, marital status, previous transplant, and complicated post-operative course did not influence employment. Favorable factors include male gender (p=0.04), younger age (<40 [p=0.0003] or <50 yr [p<0.0001]), having ≥1 dependent (p=0.04), higher education (minimum high school degree [p=0.003] or some college [p=0.002]), live donor recipient (p=0.004), wait time <2 yr (p=0.03), dialysis <2 yr (p<0.0001) or pre-dialysis (p=0.04), and pre-transplantation employment (p<0.0001). Mean time for employment was 4.9±6.3 months (median three months). Common reasons for unemployment were disability (59%) and retirement (27%). Finally, 7% correctly responded that Medicare benefits end 36 months following transplantation. CONCLUSIONS: Potentially modifiable factors to improve employment are earlier referral and better education regarding Medicare eligibility.

BK viral disease in renal transplantation.

PURPOSE OF REVIEW: BK virus is one of the most frequent causes of graft loss after renal transplantation, with BK virus-associated nephropathy occurring in roughly 8% of patients, and graft loss rates reported as high as 50%. This review is meant to highlight the literature on BK viral disease following renal transplantation published in the most recent year. RECENT FINDINGS: Prevention of BK virus-associated graft loss requires early diagnosis of BK viral replication, which is best achieved by screening for BK viral DNA in the blood. Screening intervals more frequently than the currently recommended 3 months appear to offer increased efficacy. Reduction in immunosuppression remains the mainstay for treatment of BK viral disease, with consideration given to antiviral drug therapy with leflunomide. Acute rejection may be minimized by a short course of intravenous immunoglobulin. Sirolimus appears to be a promising addition to the therapeutic armamentarium. For patients requiring re-transplantation after BK virus-associated graft loss, viral clearance from the bloodstream prior to re-transplantation should be achieved to attain optimal results. SUMMARY: BK virus is a major pathogen affecting renal allografts, although intensive surveillance and targeted dose reduction in immunosuppression with the consideration of additional antiviral drug therapy can minimize graft loss resulting from infection.

Laparoscopic living kidney donation at a single center: an examination of donor outcomes with increasing experience.

This study was undertaken to examine short-term outcomes of laparoscopic donor nephrectomy performed by transplant surgeons at a medium volume institution, with the specific goal of determining the presence of a learning curve effect. With institutional review board approval, a retrospective chart review was performed examining patient demographics, operative factors, and in-hospital outcomes over a 12-year period. Specific attention was paid to differences in outcomes between patients undergoing operation in the first versus the most recent 6-year period. Continuous and categorical variables were examined using the Wilcoxon rank sum test and χ(2) analysis, respectively. The study group consisted of 129 patients. Median operative time was 234 minutes with a median estimated blood loss of 100. The median preoperative creatinine was 0.9, with a postoperative median creatinine of 1.3. The overall complication rate was 12.4 per cent, with ileus being the most common. There were two cases of post op acute renal failure, both of which were self limited and did not require dialysis. No patients died. Patients in the most recent 6 years (n = 77) enjoyed a shorter length of stay (2 vs 3 days, P < 0.001) than patients in the first 6-year period (n = 52). Additionally, there was a trend towards decreased complications in the second era that did not reach significance (9.1% vs 17.3%, P = 0.17). Laparoscopic donor nephrectomy is an attractive means of donation, and can be performed with low risk to the donor and minimal learning curve effect.

Prophylaxis against de novo hepatitis B for liver transplantation utilizing hep B core (+) donors: does hepatitis B immunoglobulin provide a survival advantage?

Donor liver allografts with positive serology for hepatitis B core antibody [HBc (+)] have been increasingly used for liver transplantation. However, the optimal prophylactic regimen to prevent development of de novo hepatitis B has not been determined. To evaluate this, we screened United Network for Organ Sharing (UNOS) Standard Transplant Analysis and Research (STAR) registry data for adult recipients of HBc (+) organs who were HBsAg (-), and evaluated the effects of using prophylactic anti-viral therapies (HBIG and lamivudine) on patient and graft survival. Out of a total cohort of 958 patients transplanted since 2004, 61 received HBIG alone, 116 received lamivudine alone, 66 both, 509 neither and 206 were missing this information. Based on several multivariable Cox regression models, patients receiving HBIG therapy-only were observed to have a statistically significant (approximately 70%) reduction in risk of mortality compared with patients receiving lamivudine-only therapy [HR=0.29, 95% CI (0.10, 0.86), P=0.026], and a nonstatistically significant reduction in risk of graft failure. However, no graft failures were attributed to de novo hepatitis B, suggesting that any improved graft/patient survival possibly associated with HBIG therapy occurs independently of de novo hepatitis B virus (HBV) reduction. While this study cannot prove that HBIG therapy is protective for graft and patient survival after liver transplantation, these findings do highlight the need to further examine and study prophylactic use in recipients of HBc (+) donors.

Decompensated cirrhosis after renal transplantation: a case report.

Patients with end-stage liver disease with renal failure can be considered for simultaneous liver kidney transplantation. There are, however, no clear guidelines as to the management of the well-compensated cirrhotic patient with end-stage renal disease. We present the case of a patient with cirrhosis who decompensated after renal transplantation. With no indication for liver transplantation, can these patients safely undergo renal transplantation?

Perioperative anticoagulation and antiplatelet therapy in renal transplant: is there an increase in bleeding complication?

BACKGROUND: Renal transplant recipients may have comorbidities requiring anticoagulation or antiplatelet therapy. While the effects of warfarin may be neutralized with plasma infusion, those of aspirin and clopidogrel are not easily reversible and may be associated with an increased risk of bleeding. We conducted this study to evaluate the risk of bleeding complications in patients receiving perioperative anticoagulation or antiplatelet therapy. METHODS: Medical records of patients who underwent renal transplantation from July 1, 2005 to April 30, 2009 were retrospectively reviewed. Patients receiving perioperative anticoagulation or antiplatelet therapy were identified. The incidence of reoperation, transfusion utilization and decrease in serum hemoglobin from pre-operative value (ΔHgb) were compared to those on no therapy. RESULTS: Of the 327 patients identified, 105 received pre-operative anticoagulation or antiplatelet therapy, 28 received therapy post-operatively, while 213 patients received no therapy. The incidence of reoperation, transfusion utilization and ΔHgb were not significantly increased with pre-operative anticoagulation or antiplatelet therapy. With post-operative heparin infusion, the incidence of reoperation and transfusion utilization were significantly increased (p values < 0.001). Patients with activated partial thromboplastin times (aPTT) >80 s experienced significant bleeding complications. CONCLUSION: A supratherapeutic aPTT with post-operative heparin infusion was associated with the greatest risk of bleeding complication.

Review of the use of hepatitis B core antibody-positive kidney donors.

This article reviews the risks of transplanting hepatitis B core antibody (anti-HBc)-positive (+) kidneys and strategies to minimize the risks to the recipient. In the United States, there is a shortage of kidneys available for transplantation. Presently, 4% of kidneys transplanted are anti-HBc (+). In published retrospective studies, the serologic conversion rate for recipients of anti-HBc (+) kidneys varied between 0% and 27%; and the development of elevated hepatic transaminases varied between 0% and 26%. Only one published article had a trend toward increased risk of graft loss. Other published studies had no significant difference in graft or patient survival. Factors that influence the risk of transmission include hepatitis B viral load, vaccination, and antiviral therapy. If the donor is anti-HBc (+) and hepatitis B DNA negative, the risk of transmission is negligible; unfortunately, this information may not be available at the time of transplant. Vaccinated recipients with a protective hepatitis B surface antibody of at least 10 mIU/mL had a 4% conversion rate compared with 10% in recipients with antibody levels not exceeding 10 mIU/mL. Both hepatitis B immunoglobulin and lamivudine have been used in recipients of anti-HBc (+) kidneys to decrease seroconversion with success. The data do support the use of anti-HBc (+) kidneys if precautions are taken. The recipients should be informed of the risk, should be vaccinated with an adequate response, and should have surveillance serologies performed.

The role of laparoscopic donor nephrectomy in renal transplantation.

Renal transplantation is an effective treatment for patients with end-stage renal disease. Unfortunately, the number of patients waiting for transplantation greatly exceeds the number of suitable organs. Use of live kidney donors can increase the donor pool. Historically, donor nephrectomy was performed as an open technique. Its associated prolonged convalescence and long-term morbidity was likely a disincentive to donate. Laparoscopic donor nephrectomy, however, has been shown to have fewer long-term complications without compromising graft function. Since its inception, there has been an increase in the number of live donor renal transplantations performed.

Treatment of an extracapsular post-renal biopsy pseudoaneurysm with ultrasound-guided thrombin injection.

Percutaneous renal biopsy is a convenient method to obtain allograft tissue for histologic evaluation. Vascular complications, such as arteriovenous (AV) fistula and pseudoaneurysm, following renal biopsy are well known, and they usually resolve without further intervention. When symptomatic, they should be treated. We present a patient on chronic anticoagulation who developed a pseudoaneurysm after percutaneous renal biopsy. Applying techniques learned in the management of femoral artery pseudoaneurysm, we treated our patient with ultrasound-guided thrombin injection.

The analysis of metameric blue fibers and their forensic significance.

Metamerism is a phenomenon where two or more colored items with different colorant chemistries appear to the observer to be the same color. Those differences should result in different UV-visible spectra. Additionally, the literature on color science states that metameric samples will have spectra that intersect at three or more loci. Metameric samples of blue textile fibers, which were created using different coloring agents or different relative concentrations of the coloring agents, were studied to demonstrate that they could be differentiated by obtaining their spectra between 350 and 800 nm using UV-visible microspectrophotometry. However, while some of the metameric samples tested did intersect at three or more loci, others did not intersect at all. In the spectra that did intersect, no correlation was found between either the dye chemistries or the relative component concentrations.

Increasing utilization of human T-cell lymphotropic virus (+) donors in liver transplantation: is it safe?

BACKGROUND: Liver transplantation is the best treatment option for endstage liver disease. The human T-cell lymphotrophic virus (HTLV) has been associated with leukemia/lymphoma and progressive neurologic disease. There has, however, been an increased utilization of HTLV (+) grafts with little data available to support or discourage their use. METHODS: We performed univariate and multivariate analyses related to graft and patient survival for recipients of HTLV (+) donors and compared them with recipients of HTLV (-) donors using the United Network for Organ Sharing database. Complete analysis of recipient and donor clinical and demographic factors was performed. RESULTS: There were 81 adult recipients of HTLV (+) donors and 29,747 HTLV (-) donor recipients. HTLV (+) donors were more likely to be older, women, and black, with a higher average donor risk index and creatinine, and were more likely to be shared nationally. Recipients of HTLV (+) organs were at slightly elevated risk of graft failure (HR=1.39, 95% CI 0.91-2.11) and death (HR=1.20, CI 0.71-2.02) relative to HTLV (-) donor recipients (P=0.12 and 0.5, respectively). The risk decreased after multivariate analysis - graft survival (HR=1.20, CI 0.79-1.83) and patient survival (HR=1.06, CI 0.63-1.79). CONCLUSION: Our analysis reveals no statistically significant difference in graft or patient survival between recipients of HTLV (+) and (-) donors. Serious limitations of these data are that serologic testing for HTLV has a high false positive rate and that there was a short follow-up period. Until these issues are addressed, extreme caution should be exercised when using these organs.

Cecal volvulus following laparoscopic nephrectomy and renal transplantation.

Cecal volvulus is a rare cause of bowel obstruction that carries a high mortality. Recent surgery is known to be a risk factor for the development of cecal volvulus. We present a case of cecal volvulus following laparoscopic nephrectomy and renal transplantation.

Current management of sinusoidal portal hypertension.

Portal hypertension resulting from cirrhosis was one of the biggest challenges faced by general surgeons up until the past two decades. The management of portal hypertensive variceal hemorrhage has undergone dramatic changes during this period. Endoscopic variceal ligation and transjugular intrahepatic portosystemic shunts are currently used with great success. The degree of liver dysfunction remains the most important determinant of outcome in these patients. Patients with cirrhosis who have good liver function and recurrent variceal bleed remain candidates for shunt surgery. However, the need for surgical intervention has become a rarity. The success of liver transplantation has ensured that portal hypertension is cured permanently and one does not often see the critically ill and decompensated patient with cirrhosis on the surgical service. A review of the current treatment options in this very ill patient population is the primary focus of this article.