30th ESPU Congress - Lyon, France - 2019

S1: BASIC RESEARCH 1

Moderators: Magdalena Fossum (Sweden), Darius J. Bägli (Canada)

ESPU Meeting on Wednesday 24, April 2019, 13:15 - 14:15


13:15 - 13:18
S1-1 (PP)

MUTATIONAL ANALYSIS OF HOGA1 IN CHINESE PEDIATRIC PATIENTS WITH PRIMARY HYPEROXALURIA TYPE 3

Wenying WANG and Jun LI
Beijing Friendship Hospital, Capital Medical University, Urology, Beijing, CHINA

PURPOSE

The aim of our study was to analyze and characterize the mutational spectrum of PH3 in Chinese pediatric patients with kidney stones. Primary hyperoxaluria type 3(PH3) is an autosomal recessive metabolic disorder caused by inherited mutations in the HOGA1 gene. Nearly thirty HOGA1 mutations have been reported in PH3 with c.700+5G>T accounting for about 50% of the total alleles in the literatures.

MATERIAL AND METHODS

Ninety-five pediatric patients with early-onset nephrolithiasis were suspected of having PH. DNA was extracted from patient's white blood cell. The whole exome sequencing was performed. All coding regions, including exon, intron-exon boundaries, were analyzed using PCR followed by direct sequence analysis.

RESULTS

PH1, PH2 and PH3 were found in 7, 4 and 10 pediatric patients respectively. Among of PH3 patients, 8 were boys and 2 were girls, the mean age was 35 months.Three mutations not previously described in the literature about HOGA1 were identified (compound heterozygous or homozygous). The most commonly mutation was seen in 6 patients, which was a guanine to adenine substitution of the last nucleotide of exon (c.834G>A). In addition, three SNPs were found in these family (c.812G>A, c.952C>T and c.811C>T).

CONCLUSIONS

This is the largest cases reporting PH3 mutations in Asian families. Three novel HOGA1 mutations were found in associated with PH3, and c.834G>A was the most commonly seen mutation of PH3 in Chinese pediatric patients with kidney stones. Future investigation in Chinese pediatric populations, especially cases with PH3 genotype and phenotype, are highly needed.


13:18 - 13:21
S1-2 (PP)

OXIDATIVE STRESS IN PEDIATRIC UROLITHIASIS-PROTEOMIC EVIDENCE

Larisa KOVACEVIC, Hong LU, Natalija KOVACEVIC and Yegappan LAKSHMANAN
Children's Hospital of Michigan, Pediatric Urology, Detroit, USA

PURPOSE

We aimed to investigate whether oxidative stress is present in children with stones (RS) compared to healthy controls (HC) by measuring urinary proteins involved in this process.  

MATERIAL AND METHODS

Prospective quantitative proteomic comparison of pooled urine from RS (n=30, 24 females, mean age 12.95±4.03 years) versus age- and gender-matched HC (n=30), using liquid chromatography-mass spectrometry. Proteins of interest were selected using the following criteria: 1) ≥5 spectral counts; 2) ≥2-fold difference in spectral counts; and 3) ≤0.05 p-value for the Fisher’s Exact Test.

RESULTS

Of the 1813 proteins identified, 230 met the above criteria, with 163 proteins up-regulated in the RS group and 67 up-regulated in HC.

Five proteins involved in oxidative stress were over-represented in stone children.  Of those, NADPH-oxidase, a major source of reactive oxygen species, was only found in the RS group, while glutathione S-transferase A2, an important antioxidant enzyme, was more abundant in controls (Table).

Accession Number

Assigned peptides [Patient - Control]

Ratio (Patient/Control)

Fisher's Exact Test (p-Value)

BLVRB

5 - 0

Unique

0.0630

CATA

13 - 0

Unique

0.0003

SODC

35 - 13

2.69

0.0034

PERM

95 - 43

2.21

˂ 0.0001

GSTA2

19 - 38

0.59

0.0076

BLVRB-flavin reductase; CATA-catalase,SODC-Cu/Zn superoxide dismutase; PERM- myeloperoxidase; GSTA2- glutathione S-transferase A2

CONCLUSIONS

We provide proteomic evidence of oxidative stress in children with renal stones. We speculate that changes in the oxidant-antioxidant balance may cause tubular dysfunction in these patients. Targeting these proteins may have therapeutic benefits.


13:21 - 13:24
S1-3 (PP)

★ GLOBAL GENE EXPRESSION PROFILING OF BLADDER EXSTROPHY SMOOTH MUSCLE CELLS

Jason MICHAUD 1, Heather DICARLO 2 and John GEARHART 2
1) Johns Hopkins SOM, Pediatric Urology, Baltimore, USA - 2) Johns Hopkins SOM, Baltimore, USA

PURPOSE

Bladder dysfunction in patients with bladder exstrophy is characterized by an array of phenotypes, including poor bladder growth and poor detrusor contractility. To better understand smooth muscle cell physiology in bladder exstrophy, we investigated global gene expression profiles in primary bladder exstrophy smooth muscle cells using RNA sequencing technology.

MATERIAL AND METHODS

Primary human bladder smooth muscle cell lines were generated from patients with classic bladder exstrophy and controls with vesicoureteral reflux. mRNA-seq libraries were prepared from total RNA for next-generation sequencing. Reads were mapped against the human transcriptome reference (GRCh38). Normalization and differential expression analysis was performed using Partek GS 7.0, and Ingenuity Pathway Analysis (QIAGEN) was used for dataset functional analysis and modeling.

RESULTS

We identified 1422 differentially expressed genes in bladder exstrophy smooth muscle cells when compared to controls. Bladder exstrophy cell lines showed increased expression in pathways for the inflammatory response, genitourinary development and cell migration. Expression of known exstrophy candidate genes including SCL20A1, CELSR3, or the ISL-1 pathway were not altered. Inflammatory genes were upregulated in bladder exstrophy smooth muscle cells across all ages and bladder exstrophy subgroups. Furthermore, inflammatory gene expression was higher in patients undergoing delayed closure compared to newborn closure.

CONCLUSIONS

Our results represent the first application of RNA-seq technology in the study of bladder exstrophy. Bladder exstrophy smooth muscle cells demonstrated upregulation of genes relating to pathways of inflammation, genitourinary development and cell movement across all exstrophy patients. Overall, these results help further illuminate the underlying muscle pathology in exstrophy.


13:24 - 13:27
S1-4 (PP)

GENETIC VARIANTS RELATED TO SEX HORMONE BIOSYNTHESIS, GENITAL TUBERCLE, AND URETHRAL DEVELOPMENT AS A PREDICTIVE MARKER FOR HYPOSPADIAS

Jae Hyeon HAN 1, Je Sung LEE 1, Sang Hoon SONG 1, Jin-Ho CHOI 2 and Kun Suk KIM 1
1) Asan Medical Center, Department of Urology, Asan Medical Center, Seoul, REPUBLIC OF KOREA - 2) Asan Medical Center, Department of Pediatrics, Asan Medical Center, Seoul, REPUBLIC OF KOREA

PURPOSE

Hypospadias is a frequent congenital anomaly but in most cases an underlying cause has not been found. While a single gene mutation has been recognized as the factor that causes hypospadias, a wide range gene panel investigation has been conducted only in a few researches. This research will discuss about various genetic factors that influences development of hypospadias in children. 

MATERIAL AND METHODS

Twenty one hypospadias patients consisted of identical or fraternal twin and traditional patients are selected from retrospective investigation on medical records of 339 hypospadias patients of age 0 to 20 from July 2004 to May 2016. Blood sample of interest group has been collected to conduct targeted exome sequencing in an attempt to localize genetic mutation using 67 types of gene panel that influences a development of reproductive organ and testosterone and its receptor. Known and investigated genetic mutations that affects development of hypospadias are interpreted using 1000 genomes browser and potential disease causing mutations are recognized with Polyphen-2.

RESULTS

Among 21 patients, 2 genetic mutation were recognized. Heterozygous genetic mutation at AMH, BBS12, SRD5A2, BBS12 in 3 pairs of concordant twin, heterozygous genetic mutation at AMH, BBS1, WWOX, CHD7, BBS2, FGFR1 and hemizygous genetic mutation at AR, MAMLD1 were noticed. Known heterozygous FGFR1 mutation are noticed from 1 of 3 Traditional patients. No genetic mutation has been found on 4 patients. Among those mutations, CHD7, BBS1, BBS12, AMH shows great disease causing potential.

CONCLUSIONS

We found potential novel hypospadias causing genetic mutation at CHD7, BBS1, BBS12, AMH. However, another research is required to investigate influence of such mutation in a development of hypospadias.


13:27 - 13:39
Discussion
 

13:39 - 13:42
S1-5 (PP)

IN VIVO ASSESSMENT OF THE BIOMECHANICS OF RABBIT URETHRA; AN IMPORTANT AID IN OPTIMIZED TRANSLATABLE URETHRAL REGENERATIVE MEDICINE EXPERIMENTS

Tariq Osman ABBAS 1 and Cristian PENNISI 2
1) Hamad General Hospital, Pediatric Surgery, Doha, QATAR - 2) Aalborg University, Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg, DENMARK

PURPOSE

We studied the in vivo biomechanical properties of the different regions of the urethrae in a rabbit urethral reconstruction simulation model with emphasis on aging effect.

MATERIAL AND METHODS

The mechanical properties of the urethrae of different lower urinary tract segments of six 9-week-old and two 4-week-old and two 20-week-old white New Zealand male rabbits were assessed. The biomechanical parameters studied were urethral tensile strength at numerous strain values, tensile strength at break and suture retention.

RESULTS

The urethral tensile strength and Young’s modulus were significantly higher in the proximal urethra samples (p <0.001). The strain-tension curves were not significantly different between the groups.

CONCLUSIONS

All groups showed a wall tension-strain relationships in the 3 distinct sites. The urethras exhibited a decreasing ability to be distended with increasing distance from the bladder neck, indicating self-protection against damage. Rabbit urethral simulation model could be useful to test commonly used or new urethroplasty techniques and their functional (biomechanical) long-term results prior to clinical application.


13:42 - 13:45
S1-6 (PP)

★ DESIGN AND CHARACTERIZATION OF A TISSUE-ENGINEERED TRILAYER CHITOSAN, AND PLA BASED SCAFFOLD FOR URETHRAL TISSUE REPAIR.

Tariq Osman ABBAS 1 and Cristian PENNISI 2
1) Hamad General Hospital, Pediatric Surgery, Doha, QATAR - 2) Aalborg University, Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg, DENMARK

PURPOSE

Treatment of full-thickness urethral defects relies on grafts, which mimic the normal microenvironment and structure of the deficient affected tissues: epithelial, submucosal and spongiosal layers. However, the integrity and stability of the grafts are hampered by the presence of a weak interphase, generated by the layering processes of scaffold manufacturing. We hypothesized that the middle layer acts as mechanical support, being composed of a synthetic polymer like polylactide (PLA), while the other two layers performs the role of primary cell attraction sites, being constituted by high-affinity, natural matrices chitosan.

MATERIAL AND METHODS

We describe here the design and development of a trilayer graft, avoiding delamination of the three distinct layers but preserving the cues for selective generation of the unique urethral layered structure. A highly porous chitosan/polylactide/Chitosan-based grafts were obtained by layering electrospinning technique.

RESULTS

Pore structure and interconnections were designed to support the potential in vivo vascularization at the different layers. The structural integrity of the graft was successfully validated by tension-strain tests, biodegradability, suture retention and peeling tests revealing high resistance to delamination. There is a significant difference in surface wettability of Chitosan or PLA and blended scaffolds, as verified by water contact angle assays, being the chitosan layer more hydrophilic (contact angle around 35°) than the PLA-based layers (contact angle around 110°). In vitro cellular proliferation of grafts in transwell inserts was assessed, utilizing co-seeding with urothelial cells and primary bladder smooth muscle cells, that resulted in successful attachment, growth, proliferation and differentiation of these cells.

CONCLUSIONS

This easily generable skeleton of blended trilayer scaffolds are mechanically and biologically functional and forms a novel approach in tissue-engineering applications for urethral repair.


13:45 - 13:48
S1-7 (PP)

THE PATTERN OF GROWTH FACTORS GENE EXPRESSION DURING URETHRAL HEALING PROCESS IN ANIMAL MODELS

Irfan WAHYUDI 1, Chaidir Arif MOCHTAR 1, Nuryati Chairani SIREGAR 2 and Akmal TAHER 1
1) Cipto Mangunkusumo Hospital/ Faculty of Medicine, Universitas Indonesia, Urology, Jakarta Pusat, INDONESIA - 2) Cipto Mangunkusumo Hospital/ Faculty of Medicine, Universitas Indonesia, PATHOLOGY ANATOMY, Jakarta Pusat, INDONESIA

PURPOSE

Growth factors is a polypeptide that induces cell proliferation by activating a  specific membrane bound receptor. They play some roles in wound healing. The objective of this study was to observe the pattern of some key growth factors during urethral healing.

MATERIAL AND METHODS

This was an experimental study with male New Zealand rabbit as a subject, divided into 2 groups: group I as a normal urethral healing group after urethral midline dorsal incision  and group II as a urethral stricture group after deep cautherization of urethral circumference. Examination of RT PCR was done to detect and quantify  transforming growth factor (TGF) β, basic fibroblast growth factors (b-FGF), and epidermal growth factor (EGF) gene expression in specimen taken from  involved urethra.

RESULTS

Thirty two subjects survived until the end of the study. Gene expression of TGF-β was elevated during urethral healing process until day 90 in group II while reduced level of TGF-β gene expression was observed after day 60 in group I. Gene expression of  b-FGF in group I was higher than in group II in most of the time series until day 90. Increased gene expression of EGF was  demonstrated in both group during the first 3 weeks of urethral healing but almost diminished after that.   

CONCLUSIONS

This study shows some differences in gene expression of  growth factors during urethral healing process in two scenario animal model. Understanding the fluctuation of growth factors level and timing of expression can be explored to modify and improve urethral healing in the future.  


13:48 - 13:51
S1-8 (PP)

★ SINGLE NUCLEOTIDE POLYMORPHISM ARRAY ANALYSIS IN FAMILIES OF 223 CHILDREN BORN WITH ISOLATED DISTAL HYPOSPADIAS

Matthieu PEYCELON 1, Fernanda FRADE 2, Sandra CHANTOT-BASTARAUD 3, Genevieve QUENUM-MIRAILLET 3, Capucine HYON 4, Muriel HOUANG 5, Laetitia MARTINERIE 6, Annabel PAYE-JAOUEN 7, Christine GRAPIN-DAGORNO 8, Georges AUDRY 9, Serge AMSELEM 4, Marie LEGENDRE 4, Alaa EL GHONEIMI 10 and Jean-Pierre SIFFROI 11
1) AP-HP, Hôpital Universitaire Robert-Debré; Université Paris Diderot, Sorbonne Paris Cité; INSERM UMRS_933, Pediatric Urology, Reference Center for Rare Diseases (CRMR) "Malformations Rares des Voies Urinaires" (MARVU), Paris, FRANCE - 2) AP-HP, Hôpital Trousseau, Pediatric Surgery, Paris, FRANCE - 3) AP-HP, Hôpital Trousseau; INSERM UMRS_933, Genetics and Embryology, Paris, FRANCE - 4) AP-HP, Hôpital Trousseau; Sorbonne Université; INSERM UMRS_933, Genetics and Embryology, Paris, FRANCE - 5) AP-HP, Hôpital Trousseau, Endocrinology, Paris, FRANCE - 6) AP-HP, Hôpital Universitaire Robert-Debré; Université Paris Diderot, Sorbonne Paris Cité, Endocrinology, Reference Center for Rare Diseases "Maladies Endocriniennes Rares de la Croissance" (CRMERC), Paris, FRANCE - 7) AP-HP, Hôpital Universitaire Robert-Debré, Pediatric Urology, Reference Center for Rare Diseases (CRMR) "Malformations Rares des Voies Urinaires" (MARVU), Paris, FRANCE - 8) AP-HP, Hôpital Universitaire Robert-Debré; Université Paris 13, Sorbonne Paris Cité, Pediatric Urology, Reference Center for Rare Diseases (CRMR) "Malformations Rares des Voies Urinaires" (MARVU), Paris, FRANCE - 9) AP-HP, Hôpital Trousseau; Sorbonne Université, Pediatric Surgery, Paris, FRANCE - 10) AP-HP, Hôpital Universitaire Robert-Debré; Université Paris Diderot, Sorbonne Paris Cité, Pediatric Urology, Reference Center for Rare Diseases (CRMR) "Malformations Rares des Voies Urinaires" (MARVU), Paris, FRANCE - 11) AP-HP, Hôpital Trousseau; Sorbonne Université; INSERM UMRS_933, Genetics and Embryology, Plateau Mutualisé de Génétique Constitutionnelle du GHU Est, Paris, FRANCE

PURPOSE

Hypospadias is the most common malformation affecting male genitalia and its incidence is increasing. Although the causes remain often unknown, endocrine, vascular and environmental factors have been implicated. The genetic basis in case of minor forms of hypospadias is probably underestimated. The aim of this study was to perform a pangenomic study using single nucleotide polymorphism (SNP) array in children with isolated and distal hypospadias.

MATERIAL AND METHODS

A cohort of 284 children born with hypospadias has been established since 2011 of whom 223 boys had distal and isolated hypospadias. Genomic DNA was extracted from blood lymphocytes using standard techniques. A systematic karyotype was realized in all patients. SNP-array analysis was made using Cyto SNP 12 chips (Illumina®, New York, USA).

RESULTS

A total of 617 copy number variations (CNVs) were identified in this cohort of whom 192 children had known CNVs from worldwide databases. Familial study revealed a parental heredity in six cases. Twenty-five anomalies (11.2 %) that could be a potential cause of hypospadias were identified by SNP-array in this cohort of children with isolated and distal hypospadias. One case of de novo translocation 46,XY,t(7;11)(p14;q23) has been identified from a karyotype. Twenty-one patients presented CNVs which were likely to be implicated in this pathology: 16 duplications, three deletions, three double duplications, one translocation with one duplication, and one deletion and duplication encompassing a total of 22 candidate genes in 15 chromosomes. SNP-array analysis detected shared regions of homozygosity over two megabases in four patients which may harbor homozygous mutations in potential candidate genes.

CONCLUSIONS

Pangenomic analysis found surprisingly 11.2% anomalies which could be likely linked to isolated and distal hypospadias. This study highlights the usefulness of SNP-array in malformations with potential genetic causes and raises the issue of difficulties in genetic counseling.


13:51 - 13:54
S1-9 (PP)

IS IT POSSIBLE TO USE ARTIFICIAL INTELLIGENCE TOOLS TO AUTOMATIZE THE SEGMENTATION OF KIDNEY TUMORS IN CHILDREN?

Yann CHAUSSY 1, Florent MARIE 2, Thibault DELAVELLE 2, Lisa CORBAT 2, Lorédane VIEILLE 1, Marion LENOIR 3, Frédéric AUBER 1 and Julien HENRIET 2
1) CHRU Jean Minjoz, Paediatric Surgery, Besancon, FRANCE - 2) University of Bourgogne Franche-Comté, FEMTO-ST, Besançon, FRANCE - 3) CHRU Jean Minjoz, Paediatric Radiology, Besançon, FRANCE

PURPOSE

The aim of this study is to determine if is it possible to use artificial intelligence tools to automatize the segmentation of kidney tumors on the basis of CT-scan of child treated for nephroblastoma.

MATERIAL AND METHODS

We have manually segmented 10 kidney tumors using 3D Slicer software to constitute a case base. Then, we have developed a case-based reasoning system using a region growing approach for an automatic segmentation of kidneys and tumors. We have then completed the process with an adaptation phase that can modify the position of the seed (according to the expected level of grey of the structure to segment). The results of manually segmented cases and automatic segmented cases have been compared on 10 cross-sectional images using the Dice similarity Index.

RESULTS

The mean Dice similarity index was 0.92 for the segmentation of renal tumor and 0.83 for the segmentation of pathologic kidney. The automatic segmentation failed in two cases without the adaptation phase. The development of this adaptation phase permits to confirm that the seed is correctly placed before the region growing process starts.

CONCLUSIONS

Artificial intelligence tools, and particularly case-based reasoning system, are efficient to segment kidneys and renal tumors. The results could be improved by the enhancement of the case base. This system has to be developed to segment others structures like urinary cavities or renal vessels.


13:54 - 14:15
Discussion