Long-Term Functional, Cosmetic and Sexual Outcomes of Hypospadias Correction Performed in Childhood.

OBJECTIVE: Hypospadias surgery, especially when performed early in life, may have a significant impact on the urinary and sexual functions in an adult. Because the literature is still limited, this paper assesses long-term functional, cosmetic and sexual results of hypospadias repair performed in childhood. PATIENTS AND METHODS: The study includes 275 patients older than 12 years treated for a hypospadias by an Onlay, Mathieu, Duplay, or Duckett's technique between January 1990 and December 2000. Flowmetry results were retrospectively obtained from patients' charts. The Paediatric Penile Perception Score (PPPS), the Hypospadias Objective Scoring Evaluation (HOSE) and the IIEF-5 score (when older than 16 years old) questionnaires were used to assess cosmetic and sexual results. The PPPS is designed to assess both penile self-perception with regard to meatus, glans, skin and general appearance. The HOSE is a five-point scoring system designed to allow an objective appraisal of the outcome of hypospadias repair, based on evaluating meatal location, meatal shape, urinary stream, straightness of erection, and the presence and complexity of any complicating urethral fistula. RESULTS: Qmax were within age-adjusted references, independent of the surgical technique, with median (range) Qmax of 18.8 ml/s (range 3-45, n = 136). Patients expressed a high satisfaction for every single item of the penile perception scale (PPPS), with mean values between 2 (satisfied) and 3 (very satisfied). Eighty-two percent were satisfied or very satisfied of the overall evaluation of penile appearance. Eighty-one percent of patients had a normal erectile function (IIEF-5 >22; n = 35/43). CONCLUSIONS: Taking into account the limitation of a small number of patients resulting from a low 21% questionnaire's response rate, the results of this study align with previous reports from the literature and confirms that hypospadias repair using standard techniques results in acceptable functional, cosmetic and sexual outcomes. This study highlights the need of developing a set of standard approved outcomes assessments tools for evaluating the long-term impact of hypospadias repair performed in infancy.

5-Methylcytosine DNA glycosylase participates in the genome-wide loss of DNA methylation occurring during mouse myoblast differentiation.

Changes in gene expression during mouse myoblast differentiation were monitored by DNA microarray hybridisation. Four days after the onset of differentiation 2.37% of the genes increased in activity from a value of zero, whereas during the same time 1.68% of total genes had decreased expression. During the first 24 h of differentiation an average of 700 000 CpG sites per haploid genome were demethylated. Maximal loss of DNA methylation is attained after 2 days of differentiation, followed by a gradual remethylation. The highest demethylation is observed in highly repeated DNA sequences, followed by single copy sequences. When DNA replication is inhibited by aphidicolin or L-mimosine this genome-wide demethylation is still observed. During the first 3 h of differentiation there is an increase in the number of hemimethylated CpG sites, which disappear rapidly during the course of genome-wide hypomethylation. Transfection of cells with an antisense morpholino oligonucleotide to 5-methylcytosine DNA glycosylase (G/T mismatch DNA glycosylase) decreases both the activity of the enzyme and genome-wide demethylation. It is concluded that the genome-wide loss of DNA methylation in differentiating mouse myoblasts occurs in part by formation of hemimethylated CpG sites, which can serve as the substrate for 5-methylcytosine-DNA glycosylase.

5-Methylcytosine DNA glycosylase activity is also present in the human MBD4 (G/T mismatch glycosylase) and in a related avian sequence.

A 1468 bp cDNA coding for the chicken homolog of the human MBD4 G/T mismatch DNA glycosylase was isolated and sequenced. The derived amino acid sequence (416 amino acids) shows 46% identity with the human MBD4 and the conserved catalytic region at the C-terminal end (170 amino acids) has 90% identity. The non-conserved region of the avian protein has no consensus sequence for the methylated DNA binding domain. The recombinant proteins from human and chicken have G/T mismatch as well as 5-methylcytosine (5-MeC) DNA glycosylase activities. When tested by gel shift assays, human recombinant protein with or without the methylated DNA binding domain binds equally well to symmetrically, hemimethylated DNA and non-methylated DNA. However, the enzyme has only 5-MeC DNA glycosylase activity with the hemimethylated DNA. Footprinting of human MBD4 and of an N-terminal deletion mutant with partially depurinated and depyrimidinated substrate reveal a selective binding of the proteins to the modified substrate around the CpG. As for 5-MeC DNA glycosylase purified from chicken embryos, MBD4 does not use oligonucleotides containing mCpA, mCpT or mCpC as substrates. An mCpG within an A+T-rich oligonucleotide is a much better substrate than an A+T-poor sequence. The K:(m) of human MBD4 for hemimethylated DNA is approximately 10(-7) M with a V:(max) of approximately 10(-11) mol/h/microgram protein. Deletion mutations show that G/T mismatch and 5-MeC DNA glycosylase are located in the C-terminal conserved region. In sharp contrast to the 5-MeC DNA glycosylase isolated from the chicken embryo DNA demethylation complex, the two enzymatic activities of MBD4 are strongly inhibited by RNA. In situ hybridization with antisense RNA indicate that MBD4 is only located in dividing cells of differentiating embryonic tissues.

A chicken embryo protein related to the mammalian DEAD box protein p68 is tightly associated with the highly purified protein-RNA complex of 5-MeC-DNA glycosylase.

We have shown previously that DNA demethylation by chick embryo 5-methylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA. Amino acid sequences of nine peptides derived from a highly purified 5-MeC-DNA glycosylase complex were identified by Nanoelectrospray ionisation mass spectrometry to be identical to the mammalian nuclear DEAD box protein p68 RNA helicase. Antibodies directed against human p68 helicase cross-reacted with the purified 5-MeC-DNA glycosylase complex and immunoprecipitated the glycosylase activity. A 2690 bp cDNA coding for the chicken homologue of mammalian p68 was isolated and sequenced. Its derived amino acid sequence is almost identical to the human p68 DEAD box protein up to amino acid position 473 (from a total of 595). This sequence contains all the essential conserved motifs from the DEAD box proteins which are the ATPase, RNA unwinding and RNA binding motifs. The rest of the 122 amino acids in the C-terminal region rather diverge from the human p68 RNA helicase sequence. The recombinant chicken DEAD box protein expressed in Escherichia coli cross-reacts with the same p68 antibodies as the purified chicken embryo 5-MeC-DNA glycosylase complex. The recombinant protein has an RNA-dependent ATPase and an ATP-dependent helicase activity. However, in the presence or absence of RNA the recombinant protein had no 5-MeC-DNA glycosylase activity. In situ hybridisation of 5 day-old chicken embryos with antisense probes of the chicken DEAD box protein shows a high abundance of its transcripts in differentiating embryonic tissues.

A re-investigation of the ribonuclease sensitivity of a DNA demethylation reaction in chicken embryo and G8 mouse myoblasts.

Recently published results (Nucleic Acids Res. 26, 5573-5580, 1998) suggest that the ribonuclease sensitivity of the DNA demethylation reaction may be an experimental artifact due to the possible tight binding of the nucleases to the methylated DNA substrate. Using an improved protocol we show for two different systems that demethylation of hemimethylated DNA is indeed sensitive to micrococcal nuclease, requires RNA and is not an experimental artifact. The purified 5-MeC-DNA glycosylase from chicken embryos and G8 mouse myoblasts was first incubated for 5 min at 37 degrees C with micrococcal nuclease in the presence of Ca2+ in the absence of the DNA substrate. Upon blocking the nuclease activity by the addition of 25 mM EGTA, the DNA demethylation reaction was initiated by adding the labeled hemimethylated DNA substrate to the reaction mixture. Under these conditions the DNA demethylation reaction was abolished. In parallel controls, where the purified 5-MeC-DNA glycosylase was pre-incubated at 37 degrees C with the nuclease, Ca2+ and EGTA or with the nuclease and EGTA, RNA was not degraded and no inhibition of the demethylation reaction was obtained. As has already been shown for chicken embryos, the loss of 5-MeC-DNA glycosylase activity from G8 myoblasts following nuclease treatment can also be restored by the addition of synthetic RNA complementary to the methylated strand of the substrate DNA. No reactivation of 5-MeC-DNA glycosylase is obtained by complementation with a random RNA sequence, the RNA sequence complementary to the non-methylated strand or DNA, thus ruling out a non-specific competition of the RNA for the binding of the nuclease to the labeled DNA substrate.

Urethral advancement in hypospadias with a distal division of the corpus spongiosum: outcome in 158 cases.

OBJECTIVE: Outcome of urethral mobilization and advancement (Koff procedure) in hypospadias with a distal division of the corpus spongiosum and redo cases with distal urethral failure. MATERIALS AND METHODS: From January 1999 to November 2012, 158 children with a distal hypospadias (115 primary cases and 43 redo cases) underwent surgical repair using the Koff technique with a median age at surgery of 21 months (range, 12-217 months). RESULTS: Mean follow-up was 19 months (median, 14 months). Thirty patients (19%) presented with a complication (13.9% in primary cases and 32.5% in redo surgery) mostly at the beginning of our experience. Meatal stenosis was the most common one (3.5% in primary case, 6% overall). Ventral curvature (>10°), which is considered as a possible long-term iatrogenic complication of the Koff procedure, was not found in patients with fully grown penis except in one redo patient who had, retrospectively, an inadequate indication for this type of repair. Of 158 patients, 33 reached the age of puberty (>14 years old) with a mean follow-up of 34 months, only one presented with a significant ventral curvature. CONCLUSION: Urethral mobilization and advancement is a reasonable alternative for anterior hypospadias and distal fistula repair in selected cases. It has two major advantages compared to other techniques: it avoids any urethroplasty with non-urethral tissue and eliminates dysplastic tissues located beyond the division of the corpus spongiosum, which may not grow at the same pace as the rest of the penis. Significant iatrogenic curvature in fully grown penis is not supported by this series.

[Aneurysm of the arteria lusoria. Report on 4 cases]. / Aneurysma der Arteria lusoria Mitteilung von vier Fällen

Four cases of arteriosclerotic aneurysm of the arteria lusoria are described. In one patient a tumor of the superior mediastinum was suspected, the chief signs of which are dyspnea and irritative cough. In a further patient, enlargement of the left superior mediastinum was noted in routine fluoroscopy. Clinical investigation revealed an aneurysm of the arteria lusoria. In the third patient, dysphagia, slight cough and torticollis were interpreted as symptoms of a neoplasm of the esophagus or larynx. The fourth patient, in whom aneurysm of the arteria lusoria was discovered by chance at autopsy, displayed no clinical or radiological signs. Abberrant right subclavian artery (arteria lusoria) is a relatively frequent finding in autopsies. Aneurysm of the arteria lusori is apparently also a typical complication.

Physiopathological inference by computer.

A system has been programmed to allow model self-structuring to account for observations including both pathological evidence and symptomatological findings. The model modifies its structure in response to supplementary observations. The physiopathological mechanisms formalised by the model can be used for diagnostic purposes. Unknown diseases are diagnosed by direct inference of the relevant physiopathological mechanisms of the model or, if required, by automatic modification of the model that accounts for them.

5-methylcytosine-DNA glycosylase activity is present in a cloned G/T mismatch DNA glycosylase associated with the chicken embryo DNA demethylation complex.

We previously have shown that DNA demethylation by chicken embryo 5-methylcytosine DNA glycosylase (5-MCDG) needs both RNA and proteins. One of these proteins is a RNA helicase. Further peptides were sequenced, and three of them are identical to the mammalian G/T mismatch DNA glycosylase. A 3,233-bp cDNA coding for the chicken homologue of human G/T mismatch DNA glycosylase was isolated and sequenced. The derived amino acid sequence (408 aa) shows 80% identity with the human G/T mismatch DNA glycosylase, and both the C and N-terminal parts have about 50% identity. As for the highly purified chicken embryo DNA demethylation complex the recombinant protein expressed in Escherichia coli has both G/T mismatch and 5-MCDG activities. The recombinant protein has the same substrate specificity as the chicken embryo 5-MCDG where hemimethylated DNA is a better substrate than symmetrically methylated CpGs. The activity ratio of G/T mismatch and 5-MCDG is about 30:1 for the recombinant protein expressed in E. coli and 3:1 for the purified enzyme from chicken embryos. The incubation of a recombinant CpG-rich RNA isolated from the purified DNA demethylation complex with the recombinant enzyme strongly inhibits G/T mismatch glycosylase while slightly stimulating the activity of 5-MCDG. Deletion mutations indicate that G/T mismatch and 5-MCDG activities share the same areas of the N- and C-terminal parts of the protein. In reconstitution experiments RNA helicase in the presence of recombinant RNA and ATP potentiates the activity of 5-MCDG.

Overexpression of 5-methylcytosine DNA glycosylase in human embryonic kidney cells EcR293 demethylates the promoter of a hormone-regulated reporter gene.

We have shown that the DNA demethylation complex isolated from chicken embryos has a G(.)T mismatch DNA glycosylase that also possesses 5-methylcytosine DNA glycosylase (5-MCDG) activity. Herein we show that human embryonic kidney cells stably transfected with 5-MCDG cDNA linked to a cytomegalovirus promoter overexpress 5-MCDG. A 15- to 20-fold overexpression of 5-MCDG results in the specific demethylation of a stably integrated ecdysone-retinoic acid responsive enhancer-promoter linked to a beta-galactosidase reporter gene. Demethylation occurs in the absence of the ligand ponasterone A (an analogue of ecdysone). The state of methylation of the transgene was investigated by Southern blot analysis and by the bisulfite genomic sequencing reaction. Demethylation occurs downstream of the hormone response elements. No genome-wide demethylation was observed. The expression of an inactive mutant of 5-MCDG or the empty vector does not elicit any demethylation of the promoter-enhancer of the reporter gene. An increase in 5-MCDG activity does not influence the activity of DNA methyltransferase(s) when tested in vitro with a hemimethylated substrate. There is no change in the transgene copy number during selection of the clones with antibiotics. Immunoprecipitation combined with Western blot analysis showed that an antibody directed against 5-MCDG precipitates a complex containing the retinoid X receptor alpha. The association between retinoid receptor and 5-MCDG is not ligand dependent. These results suggest that a complex of the hormone receptor with 5-MCDG may target demethylation of the transgene in this system.