Shannon N. Nees

Copy-Number Disorders Are a Common Cause of Congenital Kidney Malformations

We examined the burden of large, rare, copy-number variants (CNVs) in 192 individuals with renal hypodysplasia (RHD) and replicated findings in 330 RHD cases from two independent cohorts. CNV distribution was significantly skewed toward larger gene-disrupting events in RHD cases compared to 4,733 ethnicity-matched controls (p = 4.8 × 10(-11)). This excess was attributable to known and novel (i.e., not present in any database or in the literature) genomic disorders. All together, 55/522 (10.5%) RHD cases harbored 34 distinct known genomic disorders, which were detected in only 0.2% of 13,839 population controls (p = 1.2 × 10(-58)). Another 32 (6.1%) RHD cases harbored large gene-disrupting CNVs that were absent from or extremely rare in the 13,839 population controls, identifying 38 potential novel or rare genomic disorders for this trait. Deletions at the HNF1B locus and the DiGeorge/velocardiofacial locus were most frequent. However, the majority of disorders were detected in a single individual. Genomic disorders were detected in 22.5% of individuals with multiple malformations and 14.5% of individuals with isolated urinary-tract defects; 14 individuals harbored two or more diagnostic or rare CNVs. Strikingly, the majority of the known CNV disorders detected in the RHD cohort have previous associations with developmental delay or neuropsychiatric diseases. Up to 16.6% of individuals with kidney malformations had a molecular diagnosis attributable to a copy-number disorder, suggesting kidney malformations as a sentinel manifestation of pathogenic genomic imbalances. A search for pathogenic CNVs should be considered in this population for the diagnosis of their specific genomic disorders and for the evaluation of the potential for developmental delay.

Mutations in DSTYK and Dominant Urinary Tract Malformations

BACKGROUND Congenital abnormalities of the kidney and the urinary tract are the most common cause of pediatric kidney failure. These disorders are highly heterogeneous, and the etiologic factors are poorly understood. METHODS We performed genomewide linkage analysis and whole-exome sequencing in a family with an autosomal dominant form of congenital abnormalities of the kidney or urinary tract (seven affected family members). We also performed a sequence analysis in 311 unrelated patients, as well as histologic and functional studies. RESULTS Linkage analysis identified five regions of the genome that were shared among all affected family members. Exome sequencing identified a single, rare, deleterious variant within these linkage intervals, a heterozygous splice-site mutation in the dual serine-threonine and tyrosine protein kinase gene (DSTYK). This variant, which resulted in aberrant splicing of messenger RNA, was present in all affected family members. Additional, independent DSTYK mutations, including nonsense and splice-site mutations, were detected in 7 of 311 unrelated patients. DSTYK is highly expressed in the maturing epithelia of all major organs, localizing to cell membranes. Knockdown in zebrafish resulted in developmental defects in multiple organs, which suggested loss of fibroblast growth factor (FGF) signaling. Consistent with this finding is the observation that DSTYK colocalizes with FGF receptors in the ureteric bud and metanephric mesenchyme. DSTYK knockdown in human embryonic kidney cells inhibited FGF-stimulated phosphorylation of extracellular-signal-regulated kinase (ERK), the principal signal downstream of receptor tyrosine kinases. CONCLUSIONS We detected independent DSTYK mutations in 2.3% of patients with congenital abnormalities of the kidney or urinary tract, a finding that suggests that DSTYK is a major determinant of human urinary tract development, downstream of FGF signaling. (Funded by the National Institutes of Health and others.).

Exome sequencing identified MYO1E and NEIL1 as candidate genes for human autosomal recessive steroid-resistant nephrotic syndrome

To identify gene loci associated with steroid-resistant nephrotic syndrome (SRNS), we utilized homozygosity mapping and exome sequencing in a consanguineous pedigree with three affected siblings. High-density genotyping identified three segments of homozygosity spanning 33.6 Mb on chromosomes 5, 10, and 15 containing 296 candidate genes. Exome sequencing identified two homozygous missense variants within the chromosome 15 segment; an A159P substitution in myosin 1E (MYO1E), encoding a podocyte cytoskeletal protein; and an E181K substitution in nei endonuclease VIII-like 1 (NEIL1), encoding a base-excision DNA repair enzyme. Both variants disrupt highly conserved protein sequences and were absent in public databases, 247 healthy controls, and 286 patients with nephrotic syndrome. The MYO1E A159P variant is noteworthy, as it is expected to impair ligand binding and actin interaction in the MYO1E motor domain. The predicted loss of function is consistent with the previous demonstration that Myo1e inactivation produces nephrotic syndrome in mice. Screening 71 additional patients with SRNS, however, did not identify independent NEIL1 or MYO1E mutations, suggesting larger sequencing efforts are needed to uncover which mutation is responsible for the phenotype. Our findings demonstrate the utility of exome sequencing for rapidly identifying candidate genes for human SRNS.

Proprotein convertase subtilisin/kexin type 9 potentially influences cholesterol uptake in macrophages and reverse cholesterol transport

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of low‐density lipoprotein receptor (LDLRs) molecules expressed on the cell surface. Gene inactivation of PCSK9 reduces the areas of atherosclerotic lesions in mice, and the effect is mainly dependent on LDLRs. Furthermore, a positive relationship between PCSK9 and cholesterol accumulation in the wall of the aorta has been established. However, the mechanism remains unknown. As PCSK9 is mainly expressed in atherosclerotic plaque and in the liver, we hypothesize that PCSK9 might increase oxidized LDL uptake and impair macrophage‐mediated reverse cholesterol transport, contributing to the development of atherosclerosis.

Observations on Hydroceles Following Adolescent Varicocelectomy

PURPOSE There is wide variation in the reported incidence of hydrocele after varicocelectomy (0% to 29%). We determined the incidence of hydroceles and hydrocelectomy following adolescent varicocelectomy, the time it took for them to manifest, and the results of aspiration and surgical correction. MATERIALS AND METHODS Our adolescent varicocele registry was reviewed to identify patients with a post-varicocelectomy hydrocele. We evaluated physical examination and ultrasound findings, postoperative interval to development and treatment results. RESULTS A total of 400 patients with at least 6 months of postoperative followup underwent 521 varicocelectomies (16 redo, 1 right, 104 bilateral) from 1987 to 2010. Mean followup was 32 months (range 6 to 182). Hydrocele was detected in 80 of 521 (15.4%) at a mean of 2 years after surgery. The incidence of hydrocele was higher in open vs laparoscopic (p <0.001), bilateral vs unilateral (p = 0.013), nonlymphatic sparing vs lymphatic sparing (p = 0.043) and Palomo vs laparoscopic nonlymphatic sparing (p = 0.001) procedures. Eight patients underwent aspiration for a large postoperative hydrocele. In all 8 patients fluid returned to pre-aspiration status. There were 29 patients (5.6%) who underwent Jaboulay bottleneck hydrocelectomy and none had recurrence. CONCLUSIONS Hydroceles are a common sequela of varicocelectomy, with the fewest hydroceles occurring after laparoscopic lymphatic sparing varicocelectomy. Patients should be followed for at least 2 years after varicocelectomy to examine for the presence of hydroceles. Although there have been reports on the use of aspiration for post-varicocelectomy hydrocele, we have not had success in those with a single aspiration. Jaboulay bottleneck hydrocelectomy had a 100% success rate in this select group.

Testicular Symmetry and Adolescent Varicocele—Does it Need Followup?

PURPOSE Appropriate management for adolescent varicocele with testicular symmetry is rarely discussed. We examined the natural history of varicocele in patients presenting with testicular symmetry to achieve better understanding of the clinical course. MATERIALS AND METHODS Our varicocele registry was queried for adolescent boys who presented with varicocele in association with less than 15% testicular asymmetry and who underwent at least 1 testicular asymmetry assessment 12 or more months later. Patients were stratified into 2 groups based on an initial testicular asymmetry measurement of less than 10% vs 10.0% to 14.9%. Logistic regression modeling was used to analyze the association of Tanner stage, varicocele grade, peak retrograde flow and maximum vein diameter at presentation with increased testicular asymmetry at followup. Kaplan-Meier methodology was applied to compare testicular asymmetry progression rates. RESULTS We identified 89 adolescents, of whom 52 (58.4%) and 37 (41.6%) presented with less than 10.0% and 10.0% to 14.9% testicular asymmetry, respectively. Of the patients 37 (41.6%) showed testicular asymmetry progression at a median 18-month followup. The overall 3-year testicular asymmetry progression-free rate was 48% while in patients with peak retrograde flow 30 cm per second or greater it was 23%. On multivariate analysis controlled for age, Tanner stage and varicocele grade a peak retrograde flow of 30 cm per second or greater was associated with worsening testicular asymmetry (OR 4.87, 95% CI 1.6-8.0). CONCLUSIONS Adolescents with varicocele and less than 15% testicular asymmetry are at risk for asymmetry during followup. Those with peak retrograde flow 30 cm per second or greater are at increased risk for early asymmetry while those with peak retrograde flow less than 30 cm per second may still show asymmetry but tend to do so after longer followup.

Outcomes of Targeted Treatment for Vesicoureteral Reflux in Children with Nonneurogenic Lower Urinary Tract Dysfunction

PURPOSE There is a known association between nonneurogenic lower urinary tract conditions and vesicoureteral reflux. Whether reflux is secondary to the lower urinary tract condition or coincidental is controversial. We determined the rate of reflux resolution in patients with lower urinary tract dysfunction using targeted treatment for the underlying condition. MATERIALS AND METHODS Patients diagnosed and treated for a lower urinary tract condition who had concomitant vesicoureteral reflux at or near the time of diagnosis were included. Patients underwent targeted treatment and antibiotic prophylaxis, and reflux was monitored with voiding cystourethrography or videourodynamics. RESULTS Vesicoureteral reflux was identified in 58 ureters in 36 females and 5 males with a mean age of 6.2 years. After a mean of 3.1 years of treatment reflux resolved with targeted treatment in 26 of 58 ureters (45%). All of these patients had a history of urinary tract infections before starting targeted treatment. Resolution rates of vesicoureteral reflux were similar for all reflux grades. Resolution or significant improvement of reflux was greater in the ureters of patients with dysfunctional voiding (70%) compared to those with idiopathic detrusor overactivity disorder (38%) or detrusor underutilization (40%). CONCLUSIONS Vesicoureteral reflux associated with lower urinary tract conditions resolved with targeted treatment and antibiotic prophylaxis in 45% of ureters. Unlike the resolution rates reported in patients with reflux without a coexisting lower urinary tract condition, we found that there were no differences in resolution rates among grades I to V reflux in patients with lower urinary tract conditions. Patients with dysfunctional voiding had the most improvement and greatest resolution of reflux. Additionally grade V reflux resolved in some patients.

Adolescent varicocelectomy: does artery sparing influence recurrence rate and/or catch-up growth?

The prevalence of varicocoeles is 15% in the general adolescent and adult male population and in 35–40% of men evaluated for infertility. While varicocelectomy can be performed using various methods and techniques, the laparoscopic approach allows for clear visualization of the testicular artery and lymphatics. Amongst urologists, particularly paediatric urologists, and andrologists there is much debate regarding the significance of testicular artery sparing when performing a varicocelectomy, with some believing that ligating the testicular artery impairs catch‐up growth and future fertility. On the other hand, several studies have reported higher failure rates with artery preservation. To help resolve the debate regarding the significance of artery sparing, we sought to compare varicocoele recurrence rate and catch‐up growth in patients who underwent artery sparing laparoscopic varicocelectomy compared with those who had the artery sacrificed. We identified 524 laparoscopic varicocelectomies in 425 patients from our adolescent varicocoele database. Only patients who had ultrasound determined testicular volume measurements pre‐operatively and at least 6 months post‐operatively were included. Post‐operative persistence/recurrence of varicocoele, testicular atrophy and repeat varicocelectomy were noted. Catch‐up growth was compared between procedures in those with significant pre‐operative asymmetry. Four hundred and forty primary laparoscopic varicocelectomies were performed in 355 patients (mean age: 15.5 years, range 9.3–20.6; mean follow‐up: 32.9 months, range 6.0–128.9) who had both pre‐ and post‐varicocelectomy scrotal Duplex Doppler ultrasound performed. The testicular artery was preserved in 54 varicocoeles (41 patients) and ligated in 384 varicocoeles (312 patients). We observed an increased rate of persistent/recurrent varicocoele in the artery‐sparing vs. artery ligating patients (12.2% vs. 5.4%, p = 0.09). In addition, there was no difference in catch‐up growth and no instance of testicular atrophy. As artery sparing varicocelectomy offered no advantage in regards to catch‐up growth and was associated with a higher incidence of recurrent varicocoele, preservation of the artery does not appear to be routinely necessary in adolescent varicocelectomy.

Outcomes of vesicoureteral reflux in children with non-neurogenic lower urinary tract dysfunction treated with dextranomer/hyaluronic acid copolymer (Deflux)

OBJECTIVE There has been hesitancy to use dextranomer/hyaluronic acid copolymer (DHXA, Deflux for vesicoureteral reflux (VUR) in the setting of lower urinary tract (LUT) dysfunction because of the limited number of published studies, the possibility of less success, and the manufacturers recommendations contraindicating its use in patients with active LUT dysfunction. We report on our experience using DXHA in this subset of patients whose VUR persisted despite targeted therapy for their LUT condition. MATERIALS AND METHODS We reviewed patients diagnosed with both a LUT condition and VUR who underwent subureteric DXHA while still undergoing treatment for their LUT dysfunction. Persistence of VUR was confirmed by videourodynamic studies (VUDS)/VCUG (voiding cystourethrogram) and all patients were on targeted treatment (TT) and antibiotic prophylaxis prior to and during DXHA injection. VUR was reassessed post-injection. RESULTS Fifteen patients (22 ureters; 21F,1M) met inclusion criteria (mean age 6.1 years, range 4-12). Following one to three DXHA injections, VUR resolved in 17 ureters (77%) including eight of nine ureters in dysfunctional voiding (DV) patients, five of nine in idiopathic detrusor overactivity disorder (IDOD), and four of four in detrusor underutilization disorder (DUD) patients. CONCLUSIONS DXHA is safe and effective in resolving VUR in children with associated LUT dysfunction, even before their LUT condition has fully resolved. Highest resolution rates were noted in patients with either DV or DUD or who were least symptomatic prior to injection.

Incidence, Significance and Natural History of Persistent Retrograde Venous Flow After Varicocelectomy in Children and Adolescents: Correlation with Catch-up Growth

PURPOSE Varying incidences and levels of persistent retrograde venous flow have been reported following adult and adolescent varicocelectomy but the significance remains unclear. We sought to determine the incidence and natural history of persistent flow and whether it had any effect on postoperative testicular catch-up growth. MATERIALS AND METHODS We retrospectively analyzed pre-varicocelectomy and post-varicocelectomy Doppler duplex ultrasound findings. Peak retrograde venous flow, maximum vein diameter, flow quality and varicocele grade were recorded at each visit. Catch-up growth was defined as less than 15% testicular asymmetry at final visit. RESULTS Of 330 patients (median age 15.4 years) undergoing varicocelectomy (laparoscopic in 247, open in 83) 145 had residual retrograde venous flow after Valsalva maneuver with a mean peak of 13.3 cm per second. Of 290 patients with repeat Doppler duplex ultrasound (median followup 2.6 years) 124 had initial peak retrograde venous flow less than 20 cm per second (43%) and only 17 (6%) had flow 20 cm per second or greater. Incidence of post-varicocelectomy retrograde venous flow at last visit (48%) was similar to that at initial postoperative visit (49%). Of 330 boys 20 had recurrence of palpable varicocele (grade 2 or 3), of whom 18 (90%) had initial retrograde venous flow. Catch-up growth was more likely in patients with no retrograde venous flow, and rates of catch-up growth decreased as peak retrograde venous flow increased. All 5 patients with initial testicular asymmetry and persistent retrograde venous flow at levels greater than 30 cm per second had continued testicular asymmetry (ie none had catch-up growth). CONCLUSIONS Retrograde venous flow is frequently present after varicocelectomy and is almost always associated with peak retrograde venous flow rates significantly lower than those seen in patients who are recommended for initial varicocelectomy. Retrograde venous flow tends to persist during followup at stable peak retrograde venous flow rates. Palpable recurrence and persistent testicular asymmetry are most often associated with postoperative peak retrograde venous flow rates 20 cm per second or greater.