Exome sequencing identifies novel genes and variants in patients with Hirschsprung disease.

BACKGROUND: Hirschsprung disease (HSCR) is a complex genetic disease characterized by the absence of ganglion cells in the intestines, leading to a functional obstruction in infants. At least 24 genes have been identified for the pathogenesis of HSCR. They contributed to approximately 72% of HSCR cases. We aimed to elucidate further the genetic basis of HSCR in Indonesia using the whole-exome sequencing (WES) approach. METHODS: WES was performed in 39 sporadic non-syndromic HSCR patients and 16 non-HSCR subjects as controls. Variants presented in controls were excluded, followed by in silico prediction tools and population allele frequency databases to select rare variants. We determined the minor allele frequency (MAF) using gnomAD (MAF <0.1%). RESULTS: We involved 24 (61.5%) males and 15 (38.5%) females. Most patients (62%) had short-segment aganglionosis and underwent the Duhamel procedure (41%). We identified several candidate novel variants in HSCR-related genes, including UBR4, GDNF, and ECE1. Moreover, we also identified some novel candidate genes, including a possible compound heterozygous variant in the MUTYH gene: the first variant, a known protein-truncating variant associated with colorectal cancer (CRC), p.Glu452Ter and the second variant is novel, p.Ala39Val. Moreover, the type of variants was not associated with the aganglionosis type. CONCLUSIONS: We identified several novel genes and variants, including the variant associated with CRC, that might contribute to the pathogenesis of HSCR. No genotype-phenotype associations were noted. Our study further confirms the complex network involved in enteric nervous system development and HSCR pathogenesis. LEVEL OF EVIDENCE: Level III.

Transfer factor treatment in management of peritonitis condition: An experimental study in rat.

BACKGROUND: There is a role for the immune system in improving the outcome of peritonitis cases in children. Transfer factors are one immunomodulatory treatment that can increase the activity of natural killer (NK) cells to produce interferon-gamma (IFN-γ), which is thought to increase the phagocytic activity of macrophages. This study analyzed the effects of transfer factors on the phagocytic activity of macrophages in the intraperitoneal fluid of a Wistar rat model of peritonitis. METHODS: This experimental study had a post-test-only control group design and was carried out at the Laboratory of Pharmacology and Microbiology of Padjadjaran University, Bandung, Indonesia. It analyzed the effect of transfer factors on the phagocytic activity of macrophages in the intraperitoneal fluid of Wistar rats experiencing peritonitis after being injected with Escherichia coli. An unpaired comparative t-test was performed using the SPSS program to analyze the difference between transfer factor administration and macrophage phagocytic activity levels. RESULTS: There was a statistically significant difference between the phagocytosis index values of macrophages in samples treated with transfer factors and those that were untreated (p = 0.005). CONCLUSIONS: Transfer factors increased the phagocytic activity of macrophages in a Wistar rat model of peritonitis. This suggests that transfer factors could have a role as an immunomodulatory treatment for peritonitis.

Description of mutation spectrum and polymorphism of Wilms' tumor 1 (WT1) gene in hypospadias patients in the Indonesian population.

INTRODUCTION: Hypospadias is one of the most common congenital anomalies of the penis. Previous studies reported mutation of the Wilms' tumor 1 (WT1) gene as a cause of hypospadias. The aim of this study is to describe the WT1 mutation spectrum and polymorphism in hypospadias patients in Indonesia. MATERIAL AND METHODS: DNA was isolated from 74 hypospadias patients at the Division of Pediatric Surgery, Department of Surgery Hasan Sadikin Hospital. All exons in the WT1 gene were amplified by a PCR method, followed by Sanger sequencing. Mutation analysis was performed using BioEdit software and in silico analysis using Mutation Taster, Polymorphism Phenotyping-2 (PolyPhen-2), and Sorting Intolerant from Tolerant (SIFT). RESULT: DNA analysis results showed two types of heterozygous mutations in five subjects (Table), hence the frequency of WT1 mutations was 6.7% (10/148 allele). The first mutation was a missense mutation identified in twin boys. The second was a novel heterozygous alteration in the non-coding region nine bp upstream of exon 6 (c.366-9T>C), which was identified in three patients. One heterozygous polymorphism in the coding region of exon 7 (c.471A>G/rs16754) was identified in 10 subjects. This variant did not cause any change in amino acid products (silence polymorphism). Allele frequency for the G allele (mutant allele) and A allele (wild type) was 13.5% and 86.5%, respectively. DISCUSSION: WT1 is one of the best known hypospadias genes. The WT1 gene is involved in male genital development in the early and late periods of sex determination, and hence is known as a long-term expression gene in genitalia development. Mutation analysis of WT1 in a Chinese population identified that the WT1 mutation frequency was 4.4%. The WT1 mutation frequency identified in the present study was higher, at 6.7%. Coincidentally, research subjects with p.R158H variants were monozygotic twin siblings with midshaft hypospadias accompanied by undescended testis in one and penoscrotal hypospadia with micropenis in the other. The incidence of familial hypospadias in male siblings suffering from hypospadias was reported to be 9.6% in a study conducted by Sorensen et al. Moreover, in the present study polymorphism c.471A>G(rs16754) at exon 7 was identified heterozygously in 10 research subjects (minor allele frequency 13.5%). CONCLUSION: WT1 mutations were identified in only a few cases of hypospadias and most of these were syndromic. This result implies that mutation of WT1 is not a common cause of hypospadias in the Indonesian population.

The effects of glutathione on malondialdehyde expression and seminiferous tubule damage in experimental testicular torsion-detorsion in Wistar rats.

INTRODUCTION: Testicular damage caused by ischemia reperfusion injury can be determined by malondialdehyde (MDA) expression and grading of the histopathological damage to seminiferous tubules. The aim of this study was to investigate the effect of Tationil Glutathione administration on testicular damage following experimental torsion and detorsion. METHODS: Eighteen Wistar albino rats, 5.5-6 months old and weighing 250-300 g, were divided into three equal groups. In the first group (T), torsion was created by rotating the left testis 720° in a clockwise direction and maintained for 4 h. In the T/D group, after 4 h of torsion, detorsion was performed and maintained for 3 h. In the T/D-GLUT group, we injected 25 mg iv gluthatione before performing detorsion (onset time of agent is ±5 min). RESULTS: The lowest malondialdehyde (MDA) expression was observed in the T/D-GLUT rats (P < 0.05). Grading of the histopathological damage to seminiferous tubules showed the damage to be worst in T/D and least in T/D-GLUT rats (P < 0.05). CONCLUSION: Tationil Glutathione inhibits formation of reactive oxygen species in testicular tissue during ischemia and reperfusion injury caused by experimental torsion and detorsion in Wistar rats.