Development and nationwide validation of kidney graft injury markers using urinary exosomes and microvesicles (complete English translation of the Japanese version).

BACKGROUND: Non-invasive, prompt, and proper detection tools for kidney graft injuries (KGIs) are awaited to ensure graft longevity. We screened diagnostic biomarkers for KGIs following kidney transplantation using extracellular vesicles (EVs; exosomes and microvesicles) from the urine samples of patients. METHODS: One hundred and twenty-seven kidney recipients at 11 Japanese institutions were enrolled in this study; urine samples were obtained prior to protocol/episode biopsies. EVs were isolated from urine samples, and EV RNA markers were assayed using quantitative reverse transcription polymerase chain reaction. Diagnostic performance of EV RNA markers and diagnostic formulas comprising these markers were evaluated by comparison with the corresponding pathological diagnoses. RESULTS: EV CXCL9, CXCL10, and UMOD were elevated in T-cell-mediated rejection samples compared with other KGI samples, while SPNS2 was elevated in chronic antibody-mediated rejection (cABMR) samples. A diagnostic formula developed through Sparse Logistic Regression analysis using EV RNA markers allowed us to accurately (with an area under the receiver operator characteristic curve [AUC] of 0.875) distinguish cABMR from other KGI samples. EV B4GALT1 and SPNS2 were also elevated in cABMR, and a diagnostic formula using these markers was able to distinguish between cABMR and chronic calcineurin toxicity accurately (AUC 0.886). In interstitial fibrosis and tubular atrophy (IFTA) urine samples and those with high Banff chronicity score sums (BChS), POTEM levels may reflect disease severity, and diagnostic formulas using POTEM detected IFTA (AUC 0.830) and high BChS (AUC 0.850). CONCLUSIONS: KGIs could be diagnosed with urinary EV mRNA analysis with relatively high accuracy.

Acute kidney injury caused by systemic Neisseria gonorrhoeae infection after successful kidney transplantation.

Neisseria gonorrhoeae is one of the microbes that can causes male urethritis. This microbe is most likely to be transmitted via sexual intercourse. In men, the representative infection sites are the urethra, and oral mucosa but gonococcemia is rere. We present a case of gonococcemia in a 47-year-old male successful kidney recipient. He temporarily lost his graft function due to acute kidney injury followed by sepsis; however, short-course intermittent hemodialysis and long-term intensive ceftriaxone inoculation saved his life and his graft function.

[Urinary Retention Caused by Necrotic Tissues in the Bladder of a Female Recipient of a Living-Donor Kidney Transplant : A Case Report].

We report a patient who developed urinary retention due to the presence of necrotic tissues in the bladder 5 months after kidney transplantation. The patient was a 47-year-old female who had been diagnosed with immunoglobulin A nephropathy. She requested to receive a living-donor kidney transplant from her husband, and was referred to our hospital. Given that the patient had anuria preoperatively, her bladder capacity was presumed to have decreased following the transplantation. There were no events regarding vascular anastomosis during the surgery. However, since ureteroneocystostomy was difficult to perform due to the thinning of the bladder wall, the recipient's own ureter was anastomosed to the ureter of the transplanted kidney. Since the patient had difficulty voiding soon after the indwelling urinary catheter was removed, clean intermittent self-catheterization was initiated. Abdominal computed tomography revealed perforation of the bladder and extravesical urinary leakage on postoperative day 15. An indwelling urinary catheter was reinserted as conservative treatment. We removed the indwelling urinary catheter on postoperative day 25. The patient was discharged on postoperative day 30. Five months after transplantation, the patient suddenly developed urinary retention. Cystoscopy revealed some tissue hanging from the anterior wall of the bladder. The tissue was removed, and her voiding function improved. On pathology, the tissue was found to be non-specific necrotic tissue. This finding suggested that the necrotic tissue had caused urinary retention 5 months after transplantation. The symptoms of urinary retention markedly improved after the treatment.

Cytomegalovirus peritonitis after kidney transplantation diagnosed through histopathological examination.

Among organ transplant recipients, cytomegalovirus (CMV) commonly results in various types of infection such as pneumonitis, hepatitis, and enterocolitis. However, CMV peritonitis is very rare and difficult to diagnose owing to lack of visible clinical signs. We present a case of a 35-year-old female kidney recipient who developed abdominal pain and urinary retention caused by CMV peritonitis. To our knowledge, this is the first case report of CMV peritonitis after organ transplantation to be diagnosed through histopathological examination.

Atrophic bladder in long-term dialysis patients increases the risk for urological complications after kidney transplantation.

OBJECTIVES: To evaluate the risk for urological complications after kidney transplantation at a single medical center in Japan. METHODS: In the present study, 408 kidney recipients (255 men, 153 women) were enrolled. There were 349 living and 59 deceased donors. The average age of the recipients was 42.5 ± 13.5 years, and the average pretransplant dialysis period was 71.8 ± 88.2 months. Ureteroneocystostomy was carried out on 347 patients, and ureteroureterostomy on 61 patients. We investigated the relationship between pretransplant duration of dialysis and bladder capacity, and examined the risk factors for urological complication. We also evaluated the incidence of vesicoureteral reflux in 191 recipients who underwent ureteroneocystostomy during transplantation. RESULTS: The preoperative duration of dialysis therapy showed a significant negative correlation with bladder capacity (R2  = 0.33, P < 0.001). The overall urological complication rate was 3.4% (14 patients), including urinary leakage (12 patients) and ureteral stricture (two patients). Univariate analysis showed that atrophic bladder, long-term dialysis therapy, deceased donor and ureteroureterostomy were associated with a higher incidence of urological complications (odds ratio 8.05, 4.43, 3.42 and 3.35; P < 0.01, P = 0.01, P = 0.04 and P = 0.04, respectively). Furthermore, multivariate analysis showed that atrophic bladder was the only significant factor associated with urological complications (odds ratio 10.37; P = 0.01). Among 191 recipients, vesicoureteral reflux was observed in 32 (16.8%). The incidence of vesicoureteral reflux was significantly higher in patients with atrophic bladder. CONCLUSIONS: Bladder atrophy in renal transplant recipients after long-term dialysis therapy is associated with a higher risk of urological complications.

Microvascular inflammation in early protocol biopsies of renal allografts in cases of chronic active antibody-mediated rejection.

AIM: Chronic active antibody-mediated rejection (chronic ABMR) is one important cause of late-stage renal allograft loss. However, few reports have used protocol biopsy to observe changes over time in cases that develop chronic ABMR. The aim of this study was to use protocol biopsy to clarify the histological features of cases that develop chronic ABMR. METHODS: We recruited 379 ABO compatible patients who underwent protocol biopsy at our hospital from 2010 to 2014. Seventeen of these patients were diagnosed with chronic ABMR (chronic ABMR group), and 12 patients were class 2 donor-specific antibody (DSA) positive and were not diagnosed with chronic ABMR (class 2 DSA-positive group). With the addition of a control group consisting of 30 DSA negative patients, these three groups were compared for Banff factors in protocol biopsies taken 3 months, 6 months, 1 year, 3 years, and 5 years after the transplant. RESULTS: Three months post transplant, the chronic ABMR group had a significantly higher number of patients exhibiting g + ptc > 0 than that in the control group (P = 0.01). At 1, 3, and 5 years post transplant, significantly more subjects in the chronic ABMR and class 2 DSA-positive groups compared with the control group exhibited g + ptc > 0 (P < 0.03). Five years post transplant, the chronic ABMR group exhibited a significantly higher mean c4d score than that in the control group (P = 0.02). The only significant difference observed between the chronic ABMR group and the class 2 DSA-positive group was in cg scores at 5 years post transplant, which were significantly higher in the chronic ABMR group (P = 0.03). CONCLUSIONS: These results suggest that cases exhibiting microvascular inflammation in the early post-transplant period may develop chronic ABMR, and it would be highly beneficial to perform focused electron microscope surveillance of these cases.

A TLR5 agonist inhibits acute renal ischemic failure.

Reperfusion of ischemic organs induces a potent inflammatory response initiated by the generation of reactive oxygen species that directly damage tissue and promote leukocyte infiltration and activation that also mediate tissue injury. We recently found that radiation-induced tissue injury, which is caused by radiation-induced reactive oxygen species, is attenuated by administration of CBLB502, a pharmacologically optimized derivative of the TLR5 agonist flagellin. Therefore, we tested the ability of CBLB502 to attenuate injury in a murine model of acute ischemic renal failure. CBLB502 given 30 min before imposition of bilateral renal pedicle occlusion provided marked protection against the renal dysfunction and inflammation that follows reperfusion of ischemic kidneys, including marked decreases in leukocyte infiltration, proinflammatory cytokine production, and tubular injury. Importantly, CBLB502 given within 30 min after ischemic kidney reperfusion reproduced the protective effects of pretreatment with the TLR5 agonist, indicating a window following reperfusion in which CBLB502 administration abrogates acute renal ischemic failure. Bone marrow-reconstituted chimeras were used to show that the protective effects of CBLB502 could be delivered by intact MyD88 signaling on renal parenchymal cells. Consistent with this, Ab staining of kidney sections indicated that cells lining the renal vasculature expressed TLR5. Overall, these results indicate the use of TLR5 agonists as mitigators and protectants of acute renal ischemic failure.

Tonsillectomy ameliorates histological damage of recurrent immunoglobulin A nephropathy after kidney transplantation.

AIM: Recurrence of immunoglobulin A (IgA) nephropathy (IgAN) after renal transplantation is important as a cause of graft failure under improving rejection control. However, no specific therapy for recurrent IgAN is currently available. In this study, we evaluated the histological efficacy of tonsillectomy for allograft IgAN. METHODS: Fifteen kidney recipients (male 9, female 6, mean age 40.9 ± 9.3 years), who received a diagnosis of IgA nephropathy by allograft biopsy, were enrolled in this study. Tonsillectomy was performed 44.1 ± 27.1 months after the kidney transplantation. All patients underwent a repeat graft biopsy at 23.8 ± 15.8 months after tonsillectomy. RESULTS: Six patients had microhematuria before tonsillectomy. At 12 months after treatment, the microhematuria disappeared in five of these patients and one patient had mild hematuria. Three patients had severe proteinuria (more than 1.0 g/gCr) before tonsillectomy and improved after treatment. On histological analysis, four patients had acute lesions including cellular or fibrocellular crescents. The acute lesions disappeared after these treatments in all patients. Eleven patients had chronic lesions including global sclerosis, segmental sclerosis and fibrous crescents. The chronic lesion was ameliorated in six patients, unchanged in three and deteriorated in two patients. CONCLUSIONS: Tonsillectomy improves not only clinical findings but also ameliorates histological damage caused by recurrent IgAN after kidney transplantation. Tonsillectomy is a novel and effective treatment for recurrent IgAN.

Low dose tacrolimus exposure and early steroid withdrawal with strict body weight control can improve post kidney transplant glucose tolerance in Japanese patients.

The development of diabetes mellitus (DM) after living donor kidney transplantation (KT) is a risk factor for worsening transplant kidney function, cardiac disease, and cerebrovascular disease, which may affect prognosis after KT. At our institution, all patients' glucose tolerance is evaluated perioperatively by oral glucose tolerance tests (OGTTs) at pre-KT, and 3, 6, and 12 month (mo.) after KT. We analyzed the insulinogenic index (ISI) and homeostasis model assessment beta cell (HOMA-ß) based on the immunoreactive insulin (IRI) levels to determine how glucose tolerance changed after KT in 214 patients who had not been diagnosed with DM before KT. In addition, we analyzed the body mass index (BMI) which may also influence glucose tolerance after KT. The concentration of tacrolimus (TAC) in blood was also measured as the area under the curve (AUC) to examine its effects at each sampling point. The preoperative-OGTTs showed that DM was newly diagnosed in 22 of 214 patients (10.3%) who had not been given a diagnosis of DM by the pre-KT fasting blood sugar (FBS) tests. The glucose tolerance was improved in 15 of 22 DM patients at 12 mo. after KT. ISI and IRI deteriorated only at 3 mo. after KT but improved over time. There was a trend of an inverse correlation between HOMA-ß and TAC-AUC. We also found inverse correlations between IRI and an increase in BMI from 3 to 12 mo. after KT. Early corticosteroid withdrawal or the steroid minimization protocol with tacrolimus to maintain a low level of diabetogenic tacrolimus and BMI decrease after KT used by our hospital individualizes lifestyle interventions for each patient might contribute to an improvement in post-KT glucose tolerance.

Associations Between Recipients' Feelings of Guilt for Donor and Depressive Symptoms Before Living Kidney Transplantation.

BACKGROUND: Recipient depression before kidney transplantation needs to be treated to reduce poor posttransplant outcomes. For recipients who receive living kidney transplantation, feelings of guilt for potential donors may be factors related to the presence of depressive symptoms. This study aimed to examine the association between recipients' feelings of guilt for the donor and depressive symptoms before living kidney transplantation. METHODS: Participants included 178 patients in Sapporo City General Hospital, Hokkaido, Japan, who completed a questionnaire before having living kidney transplantation from April 2009 to May 2016. Feelings of guilt for the donor, depressive symptoms using the Beck Depression Inventory-II (BDI-II), relationship to the donor, dialysis period, and socio-demographic characteristics were assessed via a questionnaire. Multivariate regression analyses were performed to examine the association between feelings of guilt for the donor and BDI-II score after multiple imputations. RESULTS: The results showed that feelings of guilt for donors were associated with depressive symptoms, especially cognitive factors. CONCLUSIONS: These findings indicate that medical staff needs to address recipients' feelings of guilt for donors before living kidney transplantation.

Preceding T-Cell-Mediated Rejection Is Associated with the Development of Chronic Active Antibody-Mediated Rejection by de Novo Donor-Specific Antibody.

AIM: Chronic active antibody-mediated rejection (CAABMR) is an important cause of late-stage renal allograft loss. Early inflammatory events such as acute rejection and infection after transplantation are considered to be the risk factors of de novo donor-specific antibody (dnDSA) production. In this study, we investigated the relationship between pre-disposing T-cell-mediated rejection and dnDSA-positive CAABMR. METHODS: We recruited 365 patients who underwent ABO-compatible renal transplantation at our hospital. Among them, 16 patients diagnosed as having dnDSA-positive CAABMR were designated as a CAABMR group, and 38 randomly selected patients were designated as a control group. All biopsies from 1 month after transplantation were included in the study. The presence or absence of borderline changes (BLCs), acute T-cell-mediated rejection (ATMR), microvascular inflammation (MVI), and C4d positive on peritubular capillaries (C4d-P) was examined. RESULTS: In the CAABMR group, BLC/ATMR was found in 12 cases (75%), and the mean duration until appearance of BLC/ATMR was 282.7 ± 328.7 days. C4d-P was found in 11 cases (68.8%), and the mean duration until its appearance was 1,432 ± 1,307 days. MVI was found in all cases, and the mean duration until its appearance was 1,333 ± 1,126 days. The mean duration until diagnosis of CAABMR was 2,268 ± 1,191 days. In the control group, BLC/ATMR was found in 13 cases (34.2%), and the mean duration until the appearance of BLC/ATMR was 173.1 ± 170.4 days. C4d-P was found in 2 cases (5.3%), and the durations until its appearance were 748 and 1,881 days. No cases of MVI were found in the control group. The frequency of BLC/ATMR was significantly higher in the CAABMR group (p < 0.01). CONCLUSION: Preceding BLC/ATMR is associated with the development of CAABMR with dnDSA.

Bladder cancer detection by urinary extracellular vesicle mRNA analysis.

OBJECTIVE: Urinary extracellular vesicles (EV) could be promising biomarkers for urological diseases. In this retrospective feasibility study, we conducted biomarker screening for early stage bladder cancer using EV mRNA analysis. METHODS: Biomarker candidates were identified through RNA-seq analysis of urinary EV from patients with non-muscle invasive bladder cancer (N=3), advanced urothelial cancer (N=3), no residual tumor after TURBT (N=2), and healthy and disease controls (N=4). Diagnostic performance was evaluated by RT-qPCR in a larger patient group including bladder cancer (N=173), renal pelvis and ureter cancer (N=33), no residual tumor and non-cancer disease control (N=36). RESULTS: Urinary EV SLC2A1, GPRC5A and KRT17 were overexpressed in pT1 and higher stage bladder cancer by 20.6-fold, 18.2-fold and 29.5-fold, respectively. These genes allowed detection of non-muscle invasive bladder cancer (AUC: 0.56 to 0.64 for pTa, 0.62 to 0.80 for pTis, and 0.82 to 0.86 for pT1) as well as pT2 and higher muscle invasive bladder cancer (AUC: 0.72 to 0.90). Subgroup analysis indicated that these markers could be useful for the detection of cytology-negative/-suspicious and recurrent bladder cancers. CONCLUSION: Three urinary EV mRNA were discovered to be elevated in bladder cancer. Urinary EV mRNA are promising biomarkers of urothelial cancer and worth further investigation.

Safety and Efficacy of Retroperitoneoscopic Living Donor Nephrectomy: Comparison of Early Complication, Donor and Recipient Outcome with Hand-Assisted Laparoscopic Living Donor Nephrectomy.

INTRODUCTION: Laparoscopic surgery has been a standard procedure of living donor nephrectomy (LDN). Transperitoneal hand-assisted laparoscopic LDN (HALDN) has been commonly reported by many centers with excellent outcome. However, there are few studies reporting retroperitoneoscopic LDN (RPLDN). MATERIALS AND METHODS: Four hundred four consecutive kidney donors (124 men, 280 women) were enrolled in this study. Age of the donors was 55.0 ± 10.7 years. RPLDN was performed for 294 donors, and HALDN for 110 donors. We compared perioperative donor outcome and early complication rates between RPLDN and HALDN to evaluate the safety and efficacy of RPLDN. RESULTS: Intraoperative blood loss was significantly less in RPLDN than in HALDN (p < 0.05). The conversion rate to open surgery was similar between the two groups. The intraoperative complication rate was 1.0% (two vascular injuries and one bowel injury) in RPLDN and 0.9% (one vascular injury) in HALDN. The postoperative complication rate was 3.4% (six surgical site infections, two postoperative bleeding, one colon perforation, one ileus, one rhabdomyolysis) in RPLDN and 1.8% (two surgical site infections) in HALDN. Although warm ischemic time was significantly longer in RPLDN than in HALDN (p < 0.01), the incidence of delayed graft function was similar between the two groups. Furthermore, there was no difference in 1-year graft survival between the two groups. CONCLUSIONS: Both RPLDN and HALDN procedures were well tolerated with minimal complication rates, and both procedures showed similar impact on recipient graft function. These results suggest that RPLDN could be a feasible option for LDN as well as HALDN.

Heat Shock Protein 90α Is a Potential Serological Biomarker of Acute Rejection after Renal Transplantation.

BACKGROUND: Heat shock protein 90 (HSP90), a molecular chaperone associated with the activation of client proteins, was recently reported to play an important role in immunologic reactions. To date, the role of HSP90 in solid organ transplantations has remained unknown. The aim of this study was to evaluate the relationship between serum HSP90α levels and acute allograft rejection after organ and tissue transplantation using serum samples from kidney allograft recipients, an in vitro antibody-mediated rejection model, and a murine skin transplantation. RESULTS: Serum HSP90α levels were significantly higher in kidney recipients at the time of acute rejection (AR) than in those with no evidence of rejection. In most cases with AR, serum HSP90 decreased to baseline after the treatment. On the other hand, serum HSP90α was not elevated as much in patients with chronic rejection, calcineurin inhibitor nephrotoxicity, or BK virus nephropathy as in AR patients. In vitro study showed that HSP90α concentration in the supernatant was significantly higher in the supernatant of human aortic endothelial cells cocultured with specific anti-HLA IgG under complement attack than in that of cells cocultured with nonspecific IgG. In mice receiving skin transplantation, serum HSP90α was elevated when the first graft was rejected and the level further increased during more severe rejection of the second graft. CONCLUSIONS: The results suggest that HSP90α is released into the serum by cell damage due to AR in organ and tissue transplantation, and it is potentially a new biomarker to help detect AR in kidney recipients.

Preservation of deep inferior epigastric artery at kidney transplantation prevents atrophy of lower rectus abdominis muscle.

BACKGROUNDS: The deep inferior epigastric artery (DIEA), which feeds the lower rectus abdominis muscle (lower RAM), is usually transected in kidney transplantation. In this study, we investigated whether preservation of DIEA can prevent lower RAM atrophy. METHODS: Two hundred and forty-five kidney transplant recipients (150 men and 95 women) were enrolled in the study (mean age 39.9 years) and were divided into two groups according to whether DIEA was transected (group A, n = 175) or preserved (group B, n = 70). The extent of lower RAM atrophy calculated in computed tomography (performed 1 year after transplantation) and incidence of lower RAM atrophy were compared between the two groups. The most predictive factors for lower RAM atrophy were assessed using a multivariate logistic regression model. RESULTS: The extent of lower RAM atrophy was significantly lower in group B (15.0 ± 18.5%) than that in group A (38.9 ± 25.4%, P = 0.003). The incidence of lower RAM atrophy was less prevalent in group B (20.0%) compared with that in group A (62.9%, P < 0.001). The sacrifice of DIEA was the only independent predictive factor for lower RAM atrophy (P < 0.001). CONCLUSIONS: Preservation of DIEA during kidney transplant can prevent lower RAM atrophy.

High renal ischemia temperature increases neutrophil chemoattractant production and tissue injury during reperfusion without an identifiable role for CD4 T cells in the injury.

Various leukocyte populations, including neutrophils and CD4 T cells, have been implicated as mediators of acute renal ischemic injury. The influence of ischemic temperature on molecular and cellular mechanisms mediating this injury was tested in a mouse model. Wild-type C57BL/6, B6.CD4(-/-), B6.CD8(-/-), and B6.RAG-1(-/-) mice subjected to bilateral renal pedicle occlusion for 30 min at a higher (37 degrees C) but not a lower (32 degrees C) ischemic maintenance temperature had clear evidence of renal dysfunction and histopathology. Ischemia imposed at the higher temperature also increased CXCL1/KC and CXCL2/MIP-2 levels and neutrophils, but not T cells or macrophages, infiltrating into the ischemic kidneys. Depletion of neutrophils but not T cells attenuated the acute ischemic injury. These results indicate the influence of ischemic temperature and time on the production of neutrophil chemoattractants and subsequent neutrophil infiltration to mediate acute ischemic injury but fail to identify a role for adaptive immune components in this injury.

Quadruple immunosuppression with basiliximab, tacrolimus, mycophenolate mofetil and prednisone is safe and effective for renal transplantation.

INTRODUCTION: Recent immunosuppression with tacrolimus and mycophenolate mofetil has improved the results of renal transplantation. In this study, we analyzed the effect and safety of basiliximab as an induction therapy. MATERIAL AND METHODS: Forty-nine kidney recipients were given tacrolimus, mycophenolate mofetil and prednisone (non-Bas group), and 31 recipients were given basiliximab as an induction therapy in addition to the triple immunosuppressants (Bas group). Graft function, incidence of acute rejection (AR), findings of protocol graft biopsy and adverse effects were compared. RESULTS: Serum creatinine within 1 yr post-transplant was comparable between the two groups. Incidence of biopsy-proven AR within 6 months post-transplant was less in the Bas group than in the non-Bas group. Borderline change at 3 months post-transplant was less in the Bas group when compared to the non-Bas group. The frequency and severity of tubulitis were higher in the non-Bas group than in the Bas group. The addition of basiliximab did not increase opportunistic infection, but reduced tacrolimus nephrotoxicity. CONCLUSION: The addition of basiliximab to the tacrolimus-based triple immunosuppressive regimen enabled us to reduce the doses of immunosuppressants and tacrolimus nephrotoxicity without increasing early rejection or infection. This regimen is safe and effective for application during the early period after renal transplantation.

Monozygotic twins with discordant sexual phenotypes due to different ratios of mosaicism of 47,X,idic(Y),idic(Y)/46,X, idic(Y)/45,X.

We report monozygotic twins of different sexual phenotypes. One of the twins had complete female external genitalia except for a mild clitoromegaly. She had bilateral gonads consisting of the wavy stroma and scant dysgenetic seminiferous tubules. No androgen secretion was induced by gonadotrophin stimulation. The other twin had hypospadiac male genitalia. His gonads were located intrascrotally and he had good androgenic response to a stimulation test. Conventional and fluorescence in situ hybridization chromosome analysis disclosed that both twins had a 47,X,idic(Y),idic(Y)/46,X,idic(Y)/45,X and 47,X, + mar x 2.ish idic(Y)(q11.2)(DYZ3++ x 2)/46,X, + mar.ish idic(Y)(q11.2)(DZY3++)/45,X. These twins were clinically monochorionic and allelotype analysis in these twins and their parents with microsatellite markers showed the affirmative probability of 0.999999994 for monozygosity. The ratio of mosaicism, gonadal histology, and testosterone productivity were reasonably correlated to the genital virilization in these monozygotic twins, showing discordant sexual phenotypes.