Alexandre Serra

In vivo effects of modification of hydroxyapatite/collagen composites with and without chondroitin sulphate on bone remodeling in the sheep tibia

The addition of chondroitin sulphate (CS) to bone cements with calcium phosphate has lead to an enhancement of bone remodeling and an increase in new bone formation in small animals. The goal of this study was to verify the effect of CS in bone cements in a large animal model simulating a clinically relevant situation of a segmental cortical defect of a critical size on bone–implant interaction and bone remodeling. The influence of adding CS to hydroxyapatite/collagen (HA/Col) composites on host response was assessed in a standard sheep tibia model. A midshaft defect of 3 cm was created in the tibiae of 14 adult female sheep. The defect was filled with a HA/Col cement cylinder in seven animals and with a CS‐modified hydroxyapatite/collagen (HA/Col/CS) cement cylinder in seven animals. In all cases the tibia was stabilized with an interlocked universal tibial nail. The animals in each group were analyzed with X‐rays, CT scans, histology, immunohistochemistry, and enzymehistochemistry, as well as histomorphometric measurements. The X‐ray investigation showed a significantly earlier callus reaction around the HA/Col/CS implants compared to HA/Col alone. The amount of newly formed bone at the end point of the experiment was significantly larger around HA/Col/CS cylinders both in the CT scan and in the histomorphometric analysis. There were still TRAP‐positive osteoclasts around the HA/Col implants after 3 months. The number of osteopontin‐positive osteoblasts and the direct bone contact were significantly higher around HA/Col/CS implants. We conclude that addition of CS enhances bone remodeling and new bone formation around HA/Col composites.

Preliminary report on elective preterm delivery at 34 weeks and primary abdominal closure for the management of gastroschisis.

INTRODUCTION We aimed to critically evaluate elective preterm delivery and immediate abdominal wall closure and other techniques for the management of gastroschisis, hypothesizing that the advantages of an elective preterm delivery outweigh possible complications related to prematurity at birth. PATIENTS AND METHODS 13 gastroschisis patients were enrolled in the elective preterm delivery program (Group 1) since 1999. Patients were delivered by cesarean section in the 34th gestational week, with immediate primary closure of the defect. Data regarding parameters at and after birth were compared with a historical control group of 10 patients conventionally managed for gastroschisis in a similar period (1994 - 1999) (Group 2). The primary endpoints of this study were the initiation of oral feeding and the length of hospital stay. RESULTS There was a significantly faster initiation of oral feeding (p = 0.0012) and a shorter hospital stay (p = 0.0160) in Group 1. The postoperative outcome was excellent in all patients. Acute and late complications were fewer and less severe in Group 1 and none were related to prematurity. CONCLUSIONS Elective preterm delivery appears to be an effective method for the management of gastroschisis, and a method whose advantages thus far have outweighed the possible complications due to prematurity.

Compound effect of PHOX2B and RET gene variants in congenital central hypoventilation syndrome combined with Hirschsprung disease

Guido Fitze,* Inke R. König, Ekkehart Paditz, Alexandre Serra, Marianne Schläfke, Dietmar Roesner, Andreas Ziegler, and Hans K. Schackert Department of Pediatric Surgery, University of Technology Dresden, Dresden, Germany Department of Pediatrics, University of Technology Dresden, Dresden, Germany Department of Surgical Research, University of Technology Dresden, Dresden, Germany Institute of Medical Biometry and Statistics, University at Lübeck, Lübeck, Germany Department of Applied Physiology, Ruhr-University Bochum, Bochum, Germany

Rare occurrence of PHOX2b mutations in sporadic neuroblastomas.

Neuroblastomas (NBs) are frequent solid tumors in childhood for which no specific genetic marker linked to their development has been identified to date. PHOX2b, which regulates the autonomic neuron development, has been associated with the development of autonomic diseases, and has been considered a potential candidate gene for neural crest-derived tumors such as NB. To ascertain the role of the PHOX2b gene in NB development, we have sequenced the complete PHOX2b coding region in tumors from 69 patients with sporadic NB, while 130 blood donors served as negative controls and 9 NB cell lines as positive controls. We found a missense deletion in exon 3 in a cell line. A further silent mutation in exon 3 (c.870C>A) was observed in 3 tumors but in none of the controls. A new polymorphism in intron 1 (IVS1-114 G>A) was observed in 31 tumor samples (44.9%) and in 68 controls (52.3%). We did not find any conclusive association of the polymorphisms or mutations in PHOX2b with the development of NB, although the large confidence intervals neither substantiate nor exclude a role for this gene in the tumor etiology.

Genomic variants in the coding region of neuronal nitric oxide synthase (NOS1) in infantile hypertrophic pyloric stenosis.

BACKGROUND Infantile hypertrophic pyloric stenosis (IHPS) is a common childhood pathology affecting 1 to 5:1000 newborns, with a genetic background suggested by familial occurrence. Neuronal nitric oxide synthase (NOS1) is a candidate gene owing to its role in the relaxation of smooth musculature and the association of the -84g>a variant of NOS1 with IHPS. METHODS We investigated NOS1 through sequencing of the complete NOS1 coding region in DNA from 43 patients with IHPS compared the genotype frequencies to 47 controls using the Cochran-Armitage trend or Fisher exact tests. RESULTS We found 19 polymorphisms in the coding region of NOS1. The variants c.3827-42_3827-43 del_insTA and c.+276 c>t were more frequent in IHPS with statistically significant exact P values (P = .010 and P = .039, respectively) yet failed to show significance after Bonferroni adjustment for multiple testing. We have also found a marginally significant occurrence of the variants c.-460A (P = .065) and c.2823+15G (P = .076). There was a significant correlation between the variants c.2706C>T ⬄ c.2823+15A>G, (r(2) = 1.00) and c.3258 C>T ⬄ c.3235+31A>G (r(2) = 1.00). CONCLUSIONS We conclude that NOS1 variants are present in patients with IHPS yet show no significant statistical association with the IHPS phenotype, suggesting at best an adjuvant role for NOS1 in IHPS.

Accuracy of corrective osteotomies in fibular malunion: a cadaver model.

Background: While incorrect length of a fibular fracture reduction can be measured by plain radiographs, accurate imaging of rotational deformities requires computed tomography (CT). Operative correction of fibular malrotation has not been accurately measured. The aim of this study was to evaluate the accuracy of operative correction of fibular malrotation. Materials and Methods: Six pairs of formalin-fixed, lower leg cadaver specimens had shortening with additional internal or external rotation induced by segmental fibular resection and plate fixation. The deformity was measured by CT. Two experienced surgeons performed standardized corrective operations on six specimens each. The postoperative results were measured by CT. Results: The mean overall accuracy for correction of malrotation was 1.58 degrees (SD = 0.8 degrees). There were no significant differences between the two surgeons performing the corrections. Conclusion: The accuracy of operative correction of malrotation in this cadaver model is in accordance with the requirements reported in clinical studies. Clinical Relevance: Considering the error margin for CT analysis, correction within 5 to 10 degrees seems practical.

Shared Copy Number Variation in Simultaneous Nephroblastoma and Neuroblastoma due to Fanconi Anemia

Concurrent emergence of nephroblastoma (Wilms Tumor; WT) and neuroblastoma (NB) is rare and mostly observed in patients with severe subtypes of Fanconi anemia (FA) with or without VACTER-L association (VL). We investigated the hypothesis that early consequences of genomic instability result in shared regions with copy number variation in different precursor cells that originate distinct embryonal tumors. We observed a newborn girl with FA and VL (aplasia of the thumbs, cloacal atresia (urogenital sinus), tethered cord at L3/L4, muscular ventricular septum defect, and horseshoe-kidney with a single ureter) who simultaneously acquired an epithelial-type WT in the left portion of the kidney and a poorly differentiated adrenal NB in infancy. A novel homozygous germline frameshift mutation in PALB2 (c.1676_c1677delAAinsG) leading to protein truncation (pGln526ArgfsX1) inherited from consanguineous parents formed the genetic basis of FA-N. Spontaneous and induced chromosomal instability was detected in the majority of cells analyzed from peripheral lymphocytes, bone marrow, and cultured fibroblasts. Bone marrow cells also showed complex chromosome rearrangements consistent with the myelodysplastic syndrome at 11 months of age. Array-comparative genomic hybridization analyses of both WT and NB showed shared gains or amplifications within the chromosomal regions 11p15.5 and 17q21.31-q25.3, including genes that are reportedly implicated in tumor development such as IGF2, H19, WT2, BIRC5, and HRAS.

Analysis of RET, ZEB2, EDN3 and GDNF Genomic Rearrangements in Central Congenital Hyperventilation Syndrome Patients by Multiplex Ligation-dependent Probe Amplification

Central congenital hypoventilation syndrome (CCHS) is an autonomous control disease producing hypoventilation, high PaCO2, and low PaO2 during quiet sleep. The main gene variants detected in CCHS are mutations in the PHOX2b gene in up to 97% of isolated cases. However, CCHS is sometimes associated with autonomic diseases such as Hirschsprung disease (HSCR). Since genomic rearrangements in particularly sensitive areas of the RET protooncogene and/or associated genes may account for the CCHS/HSCR phenotype in patients without other detectable RET variants, the aim of the present study was to identify rearrangements in the coding sequence of RET as well as in three HSCR‐associated genes (ZEB2, EDN3 and GDNF) in CCHS/HSCR patients by using Multiplex Ligation‐dependent Probe Amplification (MLPA). We have screened 27 CCHS and 11 CCHS/HSCR patients for genomic rearrangements in RET, ZEB2, EDN3 and GDNF and did not identify any deletion or amplification in these four genes in all patients. We conclude that genomic rearrangements in RET are rare and were not responsible for the CCHS/HSCR phenotype in individuals without identifiable germline RET variants in our group of patients, yet this possibility cannot be excluded altogether given the size of the cohort.

Uniparental Disomy in Somatic Mosaicism 45,X/46,XY/46,XX Associated with Ambiguous Genitalia

Disorders of sex development (DSD) affect the development of chromosomal, gonadal and/or anatomical sex. We analyzed a patient with ambiguous genitalia aiming to correlate the genetic findings with the phenotype. Blood and tissue samples from a male patient with penoscrotal hypospadias were analyzed by immunohistochemistry, karyotyping and FISH. DNA was sequenced for the AR, SRY and DHH genes, and further 26 loci in different sex chromosomes were analyzed by MLPA. The gonosomal origin was evaluated by simple tandem repeat (STR) analysis and SNP array. Histopathology revealed a streak gonad, a fallopian tube and a rudimentary uterus, positive for placental alkaline phosphatase, cytokeratin-7 and c-kit, and negative for estrogen, androgen and progesterone receptors, alpha-inhibin, alpha-1-fetoprotein, β-hCG, and oct-4. Karyotyping showed a 45,X/46,XY mosaicism, yet FISH showed both 46,XX/46,XY mosaicism (gonad and urethral plate), 46,XX (uterus and tube) and 46,XY karyotypes (rudimentary testicular tissue). DNA sequencing revealed intact sequences in SOX9, WNT4, NR0B1, NR5A1, CYP21A2, SRY, AR, and DHH. STR analysis showed only one maternal allele for all X chromosome markers (uniparental isodisomy, UPD), with a weaker SRY signal and a 4:1 ratio in the X:Y signal. Our findings suggest that the observed complex DSD phenotype is the result of somatic gonosomal mosaicism and UPD despite a normal blood karyotype. The presence of UPD warrants adequate genetic counseling for the family and frequent, lifelong, preventive follow-up controls in the patient.