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Objective: To test whether sonographically determined fecal width (SDFW) correlates with symptom improvement in a population of children with bladder and bowel dysfunction (BBD) managed with standard urotherapy (SU), even for those patients lacking initial bowel complaints., Methods: We retrospectively analyzed 200 pediatric BBD patients managed with SU for at least 3 months. Self-reported symptom improvement (complete, partial, no response) following International Children's Continence Society guidelines was tabulated. Patients with complex urologic diagnoses other than vesicoureteral reflux (VUR) were excluded. Pharmacotherapy choice, physical therapy (PT), urinary tract infection (UTI) occurrence, and VUR status were tabulated. SDFW was recorded. Non-parametric analysis of variants (ANOVA) and parametric/non-parametric t testing were used for analysis., Results: Patients had a mean age of 9.5 years (4-12). Forty-eight patients had no gastrointestinal complaints at presentation. Urotherapy yielded complete, partial, and no responses in 14% (n = 27), 33% (n = 67), and 53% (n = 106) of patients, respectively. The average SDFW for those patients with complete response (2.6 cm) was smaller than the SDFW of those with a partial response (3.1 cm) or no response (3.3 cm) (P = .0001). Non-compliance led to greater SDFW compared to compliant patients (3.7 cm and 3.1 cm, respectively, P = .0001). Fecal width was unaffected by VUR, UTI, PT, or pharmacotherapy., Conclusion: SDFW correlates well with symptom improvement in pediatric patients managed for BBD, confirming our hypothesis. SDFW is reasonable as single objective parameter to identify successful management in patients with BBD, extending to those without bowel complaints at presentation., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

To address the global burden of sickle cell disease (SCD) and the need for novel therapies, the American Society of Hematology partnered with the US Food and Drug Administration to engage the work of 7 panels of clinicians, investigators, and patients to develop consensus recommendations for clinical trial end points. The panels conducted their work through literature reviews, assessment of available evidence, and expert judgment focusing on end points related to: patient-reported outcomes (PROs), pain (non-PROs), the brain, end-organ considerations, biomarkers, measurement of cure, and low-resource settings. This article presents the findings and recommendations of the PROs, pain, and brain panels, as well as relevant findings and recommendations from the biomarkers panel. The panels identify end points, where there were supporting data, to use in clinical trials of SCD. In addition, the panels discuss where further research is needed to support the development and validation of additional clinical trial end points.

Background: Teaching and learning hypospadias repair is a major component of pediatric urology fellowship training. Educators must transfer skills to fellows, without increasing patient complications. Nevertheless, few studies report results of surgeons during their first years of independent practice., Purpose: To review outcomes of distal hypospadias repairs performed during the same 2-year period by consecutive, recently matriculated, surgeons in independent practice, and to compare them to results by their mentor (with >20 years of experience)., Materials: Exposure to hypospadias surgery during fellowship was determined from case logs of five consecutive fellows completing training from 2007-2011. TIP was the only technique used to repair distal hypospadias. No fellow operated independently or performed complete repairs under supervision. Instead, the first 3 months were spent assisting their mentor, observing surgical methodology and decision-making. Then, each performed selected portions under direct supervision, including: degloving, penile straightening, developing glans wings, incising and tubularizing the urethral plate, creating a barrier layer, sewing the glansplasty, and skin closure. Overall fellow participation in each case was <50%. In 2011-2012, urethroplasty complications (fistula, glans dehiscence, meatal stenosis, urethral stricture, diverticulum) were recorded for consecutive patients undergoing primary distal repair by these recent graduates in their independent practices. The fellow graduating in 2011 provided 1 year of data. All patients undergoing repair during the study period were included in the analysis, except those lost to follow-up after catheter removal. Composite urethroplasty complications were compared between junior surgeons, and between junior surgeons and their mentor, with Fisher's exact contingency test., Results: Training logs indicated fellow participation ranged from 76-134 hypospadias repairs, including distal, proximal and reoperative surgeries. Post-graduation case volumes ranged from 25-68 by junior surgeons versus 136 by the mentor. With similar mean follow-up, urethroplasty complication rates were statistically the same between the former fellows, and between them versus the mentor, ranging from 5-13%. Nearly all were fistulas or glans dehiscence. Junior surgeons reported they performed TIP as learned during fellowship, with one exception who used 7-0 polydioxanone rather than polyglactin for urethroplasty., Discussion: This is the first study directly comparing hypospadias surgical outcomes by recently graduated fellows in independent practice with those of their mentor. We found junior surgeons achieved similar results for distal TIP hypospadias repair. Although their participation during training largely comprised observation and surgical assistance, with discrete performance of key steps, skills sufficient to duplicate the mentor's results were transferred. These data suggest there should be no learning curve for distal hypospadias after training. This report raises several considerations for surgical educators. First, mentors should review their own results, to be certain that they are correctly performing and teaching procedures. Second, programs need to determine key steps for procedures they teach, and then emphasize their optimal performance. Finally, mentors should expect former fellows to report back their initial results of hypospadias repair to be certain lessons taught were learned. Otherwise, preventable complications resulting from technical errors will be multiplied in the children operated by their trainees as they enter independent practice., (Copyright © 2016 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Most multicystic dysplastic kidneys (MCDKs) are discovered prenatally. There is no consensus regarding initial workup and appropriate follow-up. We present a 9-year-old female who was fetally diagnosed with a MCDK and without follow-up returned with an 18-cm multicystic mass. The patient underwent laparoscopic nephrectomy. Final pathology revealed a dermoid cyst arising in a pediatric kidney, which to our knowledge has not been previously described. Patients with MCDK have hypertension as a possible sequela and possible reflux to their functioning kidney. Voiding cystourethrogram seems reasonable initially, and renal ultrasound is ideally noninvasive. Focused clinical awareness of the solitary kidney is important., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Horseshoe kidney (HSK) is the most common renal fusion anomaly. There have been reports of an association of HSKs with medical renal disease. We report a case of a child with nephrotic-range proteinuria and a HSK. As the patient was on peritoneal dialysis, the entire HSK was removed en bloc via a retroperitoneoscopic approach with early postoperative reinitiation of peritoneal dialysis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Asian nonsmoking populations have a higher incidence of lung cancer compared with their European counterparts. There is a long-standing hypothesis that the increase of lung cancer in Asian never-smokers is due to environmental factors such as second-hand smoke. We analyzed whole-genome sequencing of 30 Asian lung cancers. Unsupervised clustering of mutational signatures separated the patients into two categories of either all the never-smokers or all the smokers or ex-smokers. In addition, nearly one third of the ex-smokers and smokers classified with the never-smoker-like cluster. The somatic variant profiles of Asian lung cancers were similar to that of European origin with G.C>T.A being predominant in smokers. We found EGFR and TP53 to be the most frequently mutated genes with mutations in 50% and 27% of individuals, respectively. Among the 16 never-smokers, 69% had an EGFR mutation compared with 29% of 14 smokers/ex-smokers. Asian never-smokers had lung cancer signatures distinct from the smoker signature and their mutation profiles were similar to European never-smokers. The profiles of Asian and European smokers are also similar. Taken together, these results suggested that the same mutational mechanisms underlie the etiology for both ethnic groups. Thus, the high incidence of lung cancer in Asian never-smokers seems unlikely to be due to second-hand smoke or other carcinogens that cause oxidative DNA damage, implying that routine EGFR testing is warranted in the Asian population regardless of smoking status., (©2014 American Association for Cancer Research.)

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole-genome sequencing (WGS) study of 88 matched HCC tumor/normal pairs, 81 of which are Hepatitis B virus (HBV) positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find beta-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumor suppressor (35.2%). The Wnt/beta-catenin and JAK/STAT pathways, altered in 62.5% and 45.5% of cases, respectively, are likely to act as two major oncogenic drivers in HCC. This study also identifies several prevalent and potentially actionable mutations, including activating mutations of Janus kinase 1 (JAK1), in 9.1% of patients and provides a path toward therapeutic intervention of the disease.

Purpose: To present our initial experience with "mini" percutaneous cystolithotomy performed as an outpatient procedure in patients with neurogenic bladders., Materials & Methods: Over the last 6 years, patients with neurogenic bladders and bladder calculi were managed with outpatient percutaneous cystolithotomy. All but 1 had previously undergone appendicovesicostomy (APV) creation. The procedure was performed by first passing a pediatric cystoscope per APV. Once the calculi were visualized, and following bladder distention, additional bladder access was obtained by passage of either a 16F Peel-Away introducer using the Seldinger technique or a 5 mm laparoscopic trocar under direct vision. An ultrasonic lithotripter was then advanced through the percutaneous access site and stone fragmentation completed. Following procedure completion, a catheter was placed for 24 h for bladder decompression. All procedures were performed on an outpatient basis., Results: 12 patients underwent 18 successful operations. In 1 patient, percutaneous access was unsuccessful. Mean age at surgery was 12.3 years. Mean operative time was 72 min 8 patients had undergone previous ileocystoplasty. The remainder was rendered stone free at the completion of surgery. One patient had persistent bleeding from the intravesical trocar site necessitating fulguration and an overnight stay for observation. The remainder were sent home the same day. There were no cases of urine extravasation., Conclusions: "Mini" percutaneous cystolithotomy is a safe, effective technique for the outpatient management of bladder calculi., (Copyright © 2012. Published by Elsevier Ltd.)

The activity of metabotropic glutamate receptors (mGluRs) is known to be altered as the consequence of neurodegenerative diseases such as Alzheimer, Parkinson, and Huntington disease. However, little attention has been paid to this receptor family's potential link with cancer. Recent reports indicate altered mGluR signaling in various tumor types, and several somatic mutations in mGluR1a in lung cancer were recently described. Group 1 mGluRs (mGluR1a and mGluR5) are coupled primarily to Gαq, leading to the activation of phospholipase C and to the formation of diacylglycerol and inositol 1,4,5-trisphosphate, leading to the release of Ca(2+) from intracellular stores and protein kinase C (PKC) activation. In the present study, we investigated the intracellular localization and G protein-dependent and -independent signaling of eight GRM1 (mGluR1a) somatic mutations. Two mutants found in close proximity to the glutamate binding domain and cysteine-rich region (R375G and G396V) show both decreased cell surface expression and basal inositol phosphate (IP) formation. However, R375G shows increased ERK1/2 activation in response to quisqualate stimulation. A mutant located directly in the glutamate binding site (A168V) shows increased quisqualate-induced IP formation and, similar to R375G, increased ERK1/2 activation. Additionally, a mutation in the G protein-coupled receptor kinase 2/PKC regulatory region (R696W) shows decreased ERK1/2 activation, whereas a mutation within the Homer binding region in the carboxyl-terminal tail (P1148L) does not alter the intracellular localization of the receptor, but it induces changes in cellular morphology and exhibits reduced ERK1/2 activation. Taken together, these results suggest that mGluR1a signaling in cancer is disrupted by somatic mutations with multiple downstream consequences.

The roles of cholecystokinin 2 receptor (CCK2R) in numerous physiologic processes in the gastrointestinal tract and central nervous system are well documented. There has been some evidence that CCK2R alterations play a role in cancers, but the functional significance of these alterations for tumorigenesis is unknown. We have identified six mutations in CCK2R among a panel of 140 colorectal cancers and 44 gastric cancers. We show that these mutations increase receptor activity, activate multiple downstream signaling pathways, increase cell migration, and promote angiogenesis. Our findings suggest that somatic mutations in CCK2R may promote tumorigenesis through deregulated receptor activity and highlight the importance of evaluating CCK2R inhibitors to block both the normal and mutant forms of the receptor., (2012 AACR)

To survey hepatitis B virus (HBV) integration in liver cancer genomes, we conducted massively parallel sequencing of 81 HBV-positive and 7 HBV-negative hepatocellular carcinomas (HCCs) and adjacent normal tissues. We found that HBV integration is observed more frequently in the tumors (86.4%) than in adjacent liver tissues (30.7%). Copy-number variations (CNVs) were significantly increased at HBV breakpoint locations where chromosomal instability was likely induced. Approximately 40% of HBV breakpoints within the HBV genome were located within a 1,800-bp region where the viral enhancer, X gene and core gene are located. We also identified recurrent HBV integration events (in ≥ 4 HCCs) that were validated by RNA sequencing (RNA-seq) and Sanger sequencing at the known and putative cancer-related TERT, MLL4 and CCNE1 genes, which showed upregulated gene expression in tumor versus normal tissue. We also report evidence that suggests that the number of HBV integrations is associated with patient survival.

Purpose: While the timely diagnosis and management of pediatric torsion can lead to testicular salvage, limited data exist on rates of orchiopexy vs orchiectomy and associated factors. Thus, we examined the Pediatric Health Information System database for torsion outcomes and demographics at American pediatric hospitals., Materials and Methods: Using the Pediatric Health Information System database we performed a 7-year retrospective cohort study in children 1 to 17 years old with a primary ICD-9 diagnosis of torsion, assessing CPT codes for orchiopexy and orchiectomy. Data were analyzed with SPSS®, version 17.0., Results: Of 2,876 patients who underwent surgery for an ICD-9 diagnosis code of testicular torsion 918 (31.9%) underwent orchiectomy at a mean age of 10.7 years and 1,958 (68.1%) underwent orchiopexy at a mean age of 12.6 years (p <0.0001). In the age groups 1 to 9, 10 to 13 and 14 years or greater 274 (49.9%), 311 (29.4%) and 333 patients (26.2%), respectively, underwent orchiectomy. A higher orchiectomy rate was seen at age 1 to 9 vs 10 years or greater. Torsion and orchiectomy rates did not vary by season or geographic region. A higher orchiectomy rate was seen in black vs white children (37.6% vs 28.1%) and in patients without vs with private insurance (36.7% vs 27.0%). Multivariate analysis revealed an association of age (p <0.0001), race (p <0.0001) and insurance status (p <0.001) with orchiectomy., Conclusions: Nationally an average of 32% of the 411 pediatric torsion cases explored annually result in orchiectomy. Identified factors increasing the orchiectomy risk included age 1 to 9 years, black race and lack of private insurance. Efforts should continue to identify modifiable variables that can increase testicular salvage in patients with testicular torsion., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: A unilateral poorly or non-functioning kidney is a cause of hypertension in children. We report the outcomes of pediatric patients with unilateral renal parenchymal disease who underwent nephrectomy for hypertension., Materials and Methods: Consecutive hypertensive children undergoing nephrectomy with a unilateral poorly or non-functioning kidney were retrospectively reviewed; preoperative and postoperative clinical variables were analyzed., Results: From July 2002 to August 2009, 21 patients (8M:13F) with average age 3.5 years and average follow-up 17.8 months were studied. Eleven patients had multicystic dysplastic kidney, 8 had reflux nephropathy, and 2 had ureteropelvic junction obstruction. Fourteen of 21 (67%) had blood pressure normalization after nephrectomy. Seven of 11 with MCDK were normotensive postoperatively compared to 6/8 patients with reflux nephropathy, and 1/2 patients with UPJ obstruction. Of the 14 patients normotensive postoperatively, 7 were on antihypertensives prior to surgery. Four of 7 patients stopped their anti-hypertensive medications postoperatively, 2 decreased from 3 and 4 medications to 1, and 1 remained on an ACE inhibitor. There were 2 patients with contralateral renal scarring, both of which remained hypertensive postoperatively., Conclusions: Nephrectomy in hypertensive pediatric patients with a unilateral poorly functioning or non-functioning kidney yielded hypertension resolution in 67% (14/21), permitting cessation or diminution of antihypertensives in many patients. Given the alternative of lifelong antihypertensives with the risk of medication non-compliance and side-effects, nephrectomy is a logical option of care which can be offered to patients and families with informed knowledge of the potential for cure., (Copyright © 2011 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Purpose: We identified preoperative parameters associated with increased risk of intraoperative Wilms tumor spill., Materials and Methods: We retrospectively reviewed an institutional database of patients diagnosed with Wilms tumor between 2000 and 2008. Inclusion criteria consisted of available abdominal computerized tomogram and pathological stage I to IV disease. Patient characteristics and neoadjuvant chemotherapy use were noted. After blinding, a radiologist reviewed preoperative computerized tomogram parameters, calculating tumor volume and assigning a preoperative radiological stage., Results: Of 67 patients diagnosed with Wilms tumor 41 (22 males, 19 females) met inclusion criteria, while 26 had incomplete imaging for analysis. Comparison of patients with and without intraoperative tumor spill demonstrated no significant differences in age (3.8 vs 3.6 years), sex (3 males and 3 females vs 19 males and 16 females), body weight or tumor capsule thickness. Preoperative radiological staging was unable to predict pathological stage I to III disease. Six intraoperative tumor spills (15%) were identified (left in 4, right in 2), of which 3 were stage III disease and 3 stage IV. Without neoadjuvant chemotherapy, patients with tumors greater than 1,000 cc had an increased risk of spill (2 of 2 [100%] vs 4 of 33 [12%], p = 0.03). Of 9 patients with stage IV disease 0% (0 of 4) receiving neoadjuvant chemotherapy experienced tumor spill, while lack of neoadjuvant chemotherapy was associated with a 60% (3 of 5 patients, 1 male and 2 females) risk of stage IV spill (p = 0.17)., Conclusions: The sole significant tumor spill risk factor identifiable preoperatively was tumor volume greater than 1,000 cc. However, spill occurred at volumes less than 400 cc. Although not statistically significant, neoadjuvant chemotherapy for stage IV disease trended toward diminishing spill risk. Patients with Wilms tumors greater than 1,000 cc may benefit from neoadjuvant chemotherapy with less tumor spill, while stage IV tumors warrant further study in this regard., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Laparoscopic radical nephrectomy is emerging as the treatment of choice for localized renal malignancies in adults. Despite the widespread use of laparoscopic nephrectomy for benign renal disease in infants and children, the laparoscopic approach has not been employed for pediatric Wilms' tumor except following systemic chemotherapy. We report the results of laparoscopic radical nephrectomy for removal of unilateral Wilms' tumor prior to the administration of systemic chemotherapy in two patients.

Although the majority of colorectal cancers exhibit chromosome instability (CIN), only a few genes that might cause this phenotype have been identified and no general mechanism underlying their function has emerged. To systematically identify somatic mutations in potential CIN genes in colorectal cancers, we determined the sequence of 102 human homologues of 96 yeast CIN genes known to function in various aspects of chromosome transmission fidelity. We identified 11 somatic mutations distributed among five genes in a panel that included 132 colorectal cancers. Remarkably, all but one of these 11 mutations were in the homologs of yeast genes that regulate sister chromatid cohesion. We then demonstrated that down-regulation of such homologs resulted in chromosomal instability and chromatid cohesion defects in human cells. Finally, we showed that down-regulation or genetic disruption of the two major candidate CIN genes identified in previous studies (MRE11A and CDC4) also resulted in abnormal sister chromatid cohesion in human cells. These results suggest that defective sister chromatid cohesion as a result of somatic mutations may represent a major cause of chromosome instability in human cancers.

The elucidation of the human genome sequence has made it possible to identify genetic alterations in cancers in unprecedented detail. To begin a systematic analysis of such alterations, we determined the sequence of well-annotated human protein-coding genes in two common tumor types. Analysis of 13,023 genes in 11 breast and 11 colorectal cancers revealed that individual tumors accumulate an average of approximately 90 mutant genes but that only a subset of these contribute to the neoplastic process. Using stringent criteria to delineate this subset, we identified 189 genes (average of 11 per tumor) that were mutated at significant frequency. The vast majority of these genes were not known to be genetically altered in tumors and are predicted to affect a wide range of cellular functions, including transcription, adhesion, and invasion. These data define the genetic landscape of two human cancer types, provide new targets for diagnostic and therapeutic intervention, and open fertile avenues for basic research in tumor biology.

We present the case of a 12-year-old boy with testicular infarction secondary to Wegener's granulomatosis. Scrotal exploration revealed no evidence of testicular torsion. The testis was left in place, and, after systemic therapy, partial reperfusion was present.

DNA mismatch repair (MMR)-deficient cells typically accumulate mutations in short repetitive DNA tracts. This microsatellite instability (MSI) facilitates malignant transformation when affecting genes with growth-related and caretaker functions. To date, several putative MSI target genes have been proposed mainly based on high mutation frequency within their coding regions. However, some intronic repeat mutations have also been suggested to associate with MSI tumorigenesis, indicating the need for additional analyses on noncoding repeats. Here we have analyzed an intronic T9 repeat of semenogelin I (SEMG1) and report mutation frequencies of 51% (75 of 146) and 62% (8 of 13) in MMR-deficient primary colorectal cancers and cell lines, respectively. The putative effect of the SEMG1 mutations was assessed by RNA and protein level analyses, but no differences were detected between colorectal cancer cell lines with different SEMG1 status. Subsequently, the general background mutation frequency of MSI colorectal cancers was assessed by screening for intergenic T9 repeat alterations. One of 10 examined repeats was mutated in 70% (102 of 145) of the colorectal cancers evaluated. The frequencies observed here are notably higher than previously published in noncoding repeats shorter than 10 bp in MMR-deficient primary tumors. Our results indicate that high mutation frequencies, similar or higher than those observed in proposed and approved target genes, can be detected in repeat tracts of MSI tumors without any apparent selection pressure. These data call for urgent and thorough large-scale evaluation of mutation frequencies in neutral short repetitive sequences in MMR-deficient tumors.

Purpose: There is an increasingly large body of literature concerning tissue-engineering products that may be used in urology. Some of these are quite complex (such as multilayer patient-specific cell-seeded implants) yet the most simple and successful products to date are also the most uncomplicated: resorbable acellular extra-cellular matrices (ECMs) harvested from animals. ECMs have been used in a variety of difficult urologic reconstruction problems, and this review is intended to summarize this complex literature for the practicing urologist., Methods: Medline search of related terms such as "SIS, small intestinal submucosa, ECM, extracellular matrix, acellular matrix and urologic reconstruction". Manuscripts missed in the initial search were taken from the bibliographies of the primary references., Results: Full review of potential clinical uses of resorbable extra-cellular matrices in urologic reconstruction., Conclusions: Currently, the "state of the art" in tissue engineering solutions for urologic reconstruction means resorbable acellular xenograft matrices. They show promise when used as a pubovaginal sling or extra bolstering layers in ureteral or urethral repairs, although recent problems with inflammation following 8-ply pubovaginal sling use and failures after 1- and 4-ply SIS repair of Peyronie's disease underscore the need for research before wide adoption. Preliminary data is mixed concerning the potential for ECM urethral patch graft, and more data is needed before extended uses such as bladder augmentation and ureteral replacement are contemplated. The distant future of ECMs in urology likely will include cell-seeded grafts with the eventual hope of producing "off the shelf" replacement materials. Until that day arrives, ECMs only fulfill some of the requirements for the reconstructive urologist.

Tumor endothelial marker (TEM)8 was uncovered as a gene expressed predominantly in tumor endothelium, and its protein product was recently identified as the receptor for anthrax toxin. Here, we demonstrate that TEM8 protein is preferentially expressed in endothelial cells of neoplastic tissue. We used the extracellular domain of TEM8 to search for ligands and identified the alpha 3 subunit of collagen VI as an interacting partner. The TEM8-interacting region on collagen alpha 3(VI) was mapped to its COOH-terminal C5 domain. Remarkably, collagen alpha 3(VI) is also preferentially expressed in tumor endothelium in a pattern concordant with that of TEM8. These results suggest that the TEM8/C5 interaction may play an important biological role in tumor angiogenesis.

PAX3 is a transcription factor important for neural, muscle, and facial development in vertebrates. To identify genes regulated by PAX3, we used a cyclic amplification and selection of targets (CASTing) strategy to isolate cis-regulatory elements bound by PAX3. CASTing libraries were constructed with mouse DNA fragments bound by mouse PAX3, and human genomic DNA fragments bound by human PAX3 and the fusion protein PAX3-FKHR. Approximately 1000 clones were sequenced from each of these three libraries. Numerous putative targets of PAX3 and PAX3-FKHR were identified and six genes, Itm2A, Fath, FLT1, TGFA, BVES, and EN2, were examined closely. The genomic DNA fragments near these genes contain PAX3 binding sites and confer PAX3-dependent regulation. The expression levels of these genes correlate with the PAX3 expression levels in mouse embryos or with PAX3-FKHR expression levels in rhabdomyosarcoma cell lines, and indicate they may be part of the PAX3 regulatory circuitry during embryogenesis and tumor formation.

B-Raf contains multiple Akt consensus sites located within its amino-terminal regulatory domain. One site, Ser(364), is conserved with c-Raf but two additional sites, Ser(428) and Thr(439), are unique to B-Raf. We have investigated the role of both the conserved and unique phosphorylation sites in the regulation of B-Raf activity in vitro and in vivo. We show that phosphorylation of B-Raf by Akt occurs at multiple residues within its amino-terminal regulatory domain, at both the conserved and unique phosphorylation sites. The alteration of the serine residues within the Akt consensus sites to alanines results in a progressive increase in enzymatic activity in vitro and in vivo. Furthermore, expression of Akt inhibits epidermal growth factor-induced B-Raf activity and inhibition of Akt with LY294002 up-regulates B-Raf activity, suggesting that Akt negatively regulates B-Raf in vivo. Our results demonstrate that B-Raf activity can be negatively regulated by Akt through phosphorylation in the amino-terminal regulatory domain of B-Raf. This cross-talk between the B-Raf and Akt serine/threonine kinases is likely to play an important role in modulating the signaling specificity of the Ras/Raf pathway and in promoting biological outcome.

Mutations in myosin XV are responsible for congenital profound deafness DFNB3 in humans and deafness and vestibular defects in shaker 2 mice. By combining direct cDNA analyses with a comparison of 95.2 kb of genomic DNA sequence from human chromosome 17p11.2 and 88.4 kb from the homologous region on mouse chromosome 11, we have determined the genomic and mRNA structures of the human (MYO15) and mouse (Myo15) myosin XV genes. Our results indicate that full-length myosin XV transcripts contain 66 exons, are >12 kb in length, and encode 365-kDa proteins that are unique among myosins in possessing very long approximately 1200-aa N-terminal extensions preceding their conserved motor domains. The tail regions of the myosin XV proteins contain two MyTH4 domains, two regions with similarity to the membrane attachment FERM domain, and a putative SH3 domain. Northern and dot blot analyses revealed that myosin XV is expressed in the pituitary gland in both humans and mice. Myosin XV transcripts were also observed by in situ hybridization within areas corresponding to the sensory epithelia of the cochlea and vestibular systems in the developing mouse inner ear. Immunostaining of adult mouse organ of Corti revealed that myosin XV protein is concentrated within the cuticular plate and stereocilia of cochlear sensory hair cells. These results indicate a likely role for myosin XV in the formation or maintenance of the unique actin-rich structures of inner ear sensory hair cells., (Copyright 1999 Academic Press.)

PAX3 is a member of the paired box family of transcription factors that function during embryogenesis and cancer epigenesis. Mutations in PAX3 cause Waardenburg syndrome (types 1 and 3), Craniofacial-deafness-hand syndrome and alveolar rhabdomyosarcoma in humans and the Splotch phenotype in mice. In this study, we describe the genomic structure of PAX3, including novel coding sequences and the complete 3' UTR. Alternative transcripts of PAX3 were identified in various tissues, including human adult skeletal muscle and mouse embryos. One of the novel alternative transcripts is evolutionarily conserved in quail and can transactivate a reporter construct containing the mouse c-met promoter. The sequences and alternative transcripts reported herein extend our understanding of the function and evolution of PAX3 in vertebrates and enable a comprehensive mutation screen for individuals with Waardenburg syndrome.

Human C8 is one of five components of the membrane attack complex of complement. It is an oligomeric protein composed of three subunits (C8 alpha, C8 beta, and C8 gamma) that are derived from different genes. C8 alpha and C8 beta are homologous and both contain a pair of tandemly arranged N-terminal modules [thrombospondin type 1 (TSP1) + low-density lipoprotein receptor class A (LDLRA)], an extended middle segment referred to as the membrane attack complex/perforin region (MACPF), and a pair of C-terminal modules [epidermal growth factor (EGF) + TSP1]. During biosynthetic processing, C8 alpha and C8 gamma associate to form a disulfide-linked dimer (C8 alpha-gamma) that binds to C8 beta through a site located on C8 alpha. In this study, the location of binding sites for C8 beta and C8 gamma and the importance of the modules in these interactions were investigated by use of chimeric and truncated forms of C8 alpha in which module pairs were either exchanged for those in C8 beta or deleted. Results show that exchange or deletion of one or both pairs of modules does not abrogate the ability of C8 alpha to form a disulfide-linked dimer when coexpressed with C8 gamma in COS cells. Furthermore, each chimeric and truncated form of C8 alpha-gamma retains the ability to bind C8 beta; however, only those containing the TSP1 + LDLRA modules from C8 alpha are hemolytically active. These results indicate that binding sites for C8 beta and C8 gamma reside within the MACPF region of C8 alpha and that interaction with either subunit is not dependent on the modules. They also suggest that the N-terminal modules in C8 alpha are important for C9 binding and/or expression of C8 activity.

The nonsyndromic congenital recessive deafness gene, DFNB3, first identified in Bengkala, Bali, was mapped to a approximately 12-cM interval on chromosome 17. New short tandem repeats (STRs) and additional DNA samples were used to identify recombinants that constrain the DFNB3 interval to less, similar6 cM on 17p11.2. Affected individuals from Bengkala and affected members of a family with hereditary deafness who were from Bila, a village neighboring Bengkala, were homozygous for the same alleles for six adjacent STRs in the DFNB3 region and were heterozygous for other distal markers, thus limiting DFNB3 to an approximately 3-cM interval. Nonsyndromic deafness segregating in two unrelated consanguineous Indian families, M21 and I-1924, were also linked to the DFNB3 region. Haplotype analysis indicates that the DFNB3 mutations in the three pedigrees most likely arose independently and suggests that DFNB3 makes a significant contribution to hereditary deafness worldwide. On the basis of conserved synteny, mouse deafness mutations shaker-2 (sh2) and sh2J are proposed as models of DFNB3. Genetic mapping has refined sh2 to a 0.6-cM interval of chromosome 11. Three homologous genes map within the sh2 and DFNB3 intervals, suggesting that sh2 is the homologue of DFNB3.

Two percent of the residents of Bengkala, Bali, have profound, congenital, neurosensory, nonsyndromal deafness due to an autosomal recessive mutation at the DFNB3 locus. We have employed a direct genome-wide disequilibrium search strategy, allele-frequency-dependent homozygosity mapping (AHM), and an analysis of historical recombinants to map DFNB3 and position the locus relative to flanking markers. DFNB3 maps to chromosome 17, closest to D17S261, pRM7-GT and D17S805. In individuals homozygous for DFNB3, historical recombinant genotypes for the flanking markers, D17S122 and D17S783, place DFNB3 in a 5.3 cM interval of the pericentromeric region of chromosome 17 on a refined linkage map of 17p-17q12. Based on conserved synteny, the murine sh2 gene may be the homologue of DFNB3.