nan Gunadi

Effects of RET and NRG1 polymorphisms in Indonesian patients with Hirschsprung disease

BACKGROUNDnHirschsprung disease (HSCR) is a neurocristopathy characterized by absence of intramural ganglion cells along variable lengths of the gastrointestinal tract in neonates. Three polymorphisms, rs2435357, within a conserved transcriptional enhancer of RET, and, rs7835688 and rs16879552, within intron 1 of NRG1, have been shown to be associated with isolated forms of HSCR. We wished to replicate these findings, and study the interactions between these variants, in Indonesian HSCR patients.nnnMETHODSnSixty isolated HSCR patients and 124 controls were ascertained for this study. The three genetic markers were examined using TaqMan Genotyping Assays in genomic DNA for association studies.nnnRESULTSnRET rs2435357 showed the strongest association with HSCR both by case-control analysis (p=2.5 × 10(-8)) and transmission disequilibrium test (p=4.2 × 10(-6)). NRG1 rs7835688 was modestly associated with HSCR only by case-control analysis (p=4.3 × 10(-3)), whereas rs16879552 demonstrated no association (p>0.097). Two locus analyses of variants showed significant interactions with increased and decreased disease risks of HSCR at NRG1 but conditional on rs2435357 genotype.nnnCONCLUSIONSnRET and NRG1 variants are common susceptibility factors for HSCR in Indonesia. These common variants demonstrate that development of HSCR requires joint effects of RET and NRG1 early in gut development.

Two novel mutations in the ED1 gene in Japanese families with X-linked hypohidrotic ectodermal dysplasia.

X-linked hypohidrotic ectodermal dysplasia (XLHED), which is characterized by hypodontia, hypotrichosis, and hypohidrosis, is caused by mutations in ED1, the gene encoding ectodysplasin-A (EDA). This protein belongs to the tumor necrosis factor ligand superfamily. We analyzed ED1 in two Japanese patients with XLHED. In patient 1, we identified a 4-nucleotide insertion, c.119-120insTGTG, in exon 1, which led to a frameshift mutation starting from that point (p.L40fsX100). The patients mother was heterozygous for this mutation. In patient 2, we identified a novel missense mutation, c.1141G>C, in exon 9, which led to a substitution of glycine with arginine in the TNFL domain of EDA (p.G381R). This patients mother and siblings showed neither symptoms nor ED1 mutations, so this mutation was believed to be a de novo mutation in maternal germline cells. According to molecular simulation analysis of protein structure and electrostatic surface, p.G381R increases the distance between K375 in monomer A and K327 in monomer B, which suggests an alteration of overall structure of EDA. Thus, we identified two novel mutations, p.L40fsX100 and p.G381R, in ED1 of two XLHED patients. Simulation analysis suggested that the p.G381R mutation hampers binding of EDA to its receptor via alteration of overall EDA structure.

Effects of SEMA3 polymorphisms in Hirschsprung disease patients

PurposeRecently, genetic markers within a locus on 7q21.11 containing the SEMA3A, SEMA3C, and SEMA3D genes were reported to be associated with Hirschsprung disease (HSCR). Here, we investigated three polymorphisms, rs1583147, rs12707682, and rs11766001, at this locus to determine their potential contributions to the susceptibility of Indonesian HSCR patients.MethodsThree variants were analyzed in 60 non-syndromic HSCR patients and 118 ethnicity-matched controls for association studies by genotyping.ResultsThe risk allele frequencies of SEMA3 rs12707682 (allele C) and rs1583147 (allele T) is higher in cases, 53 and 23xa0%, than in controls, at 42 and 13xa0%, respectively. However, these frequency differences were not statistically significant with p value of 0.06 and 0.023, respectively. These findings were consistent with transmission disequilibrium test results with p values of 0.041 and 0.11 for rs12707682 and rs1583147, respectively. Furthermore, the frequencies of SEMA3 rs11766001 risk allele in HSCR cases and controls were 1.7 and 0.8xa0%, respectively.ConclusionsSEMA3 rs12707682 and rs1583147 variants are not common risk factors for HSCR in Indonesia. The rarity of the SEMA3 rs11766001 polymorphism in Indonesian population might be due to a founder effect.

Comparison of Hirschsprung-associated enterocolitis following Soave and Duhamel procedures

BACKGROUNDnHirschsprung-associated enterocolitis (HAEC) represents the primary cause of high morbidity and mortality in Hirschsprung disease (HSCR) patients. The most common surgical methods for HSCR are the Soave and Duhamel procedures. Therefore, we aimed to compare the HAEC frequency following the Soave and Duhamel procedures.nnnMETHODSnMedical records were retrospectively analyzed for patients who underwent the Soave and Duhamel pull-through at Dr. Sardjito Hospital, Indonesia from 2010 to 2015. The diagnosis of HAEC was determined using a HAEC scoring system.nnnRESULTSnOne hundred patients were involved (Soave: 52 males and 19 females vs. Duhamel: 23 males and 6 females, p=0.62). There was significant difference in mean age at pull-through (Soave: 29.9±45.2 vs. Duhamel: 50.8±47.5months, p=0.04), whereas mean age of HSCR diagnosis and pre-operative enterocolitis frequency did not differ significantly between groups (Soave: 25.4±41.0 vs. Duhamel: 43.7±48.1months, p=0.06, and Soave: 7% vs. Duhamel: 14%, p=0.44, respectively). The HAEC frequency after pull-through was significantly higher in the Duhamel than the Soave group (28% vs. 10%, respectively, p=0.03). Furthermore, pre-operative enterocolitis showed a significant association with HAEC following pull-through (p=2.0×10-4) and the risk of HAEC after Soave pull-through was increased in long-segment aganglionosis compared to short-segment HSCR (p=0.015).nnnCONCLUSIONSnThe frequency of HAEC was significantly higher after the Duhamel than the Soave procedure. Moreover, patients with pre-operative enterocolitis are prone to have HAEC following pull-through.nnnLEVEL OF EVIDENCEnIII.

The utility of the hematoxylin and eosin staining in patients with suspected Hirschsprung disease

BackgroundWhile immunohistochemistry (IHC) methods have been widely conducted for the diagnosis of Hirschsprung disease (HSCR) in developed countries, there are very few studies on their use in developing countries where hematoxylin and eosin (HE) staining is a key element of the diagnosis of HSCR. We aimed to determine the accuracy of HE staining in the diagnosis of HSCR using S100 IHC as the reference standard in Indonesia.MethodsAll histopathology performed for the suspicion of HSCR patients from January 2013 to August 2015 in Dr. Sardjito Hospital, Yogyakarta, Indonesia, were retrospectively reviewed.ResultsOur study included 23 HSCR patients: 9 males and 14 females. The HE staining revealed 14 negative (absence of ganglion cells) cases (61%) and 9 positive (presence of ganglion cells) cases (39%). In S100 IHC, out of the 9 positive cases by HE staining, 6 (67%) were confirmed for having ganglion cells; and out of the 14 negative cases by HE staining, 12 (86%) were reported negative and 2 (14%) were positive by S100 IHC staining. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy rates of the HE staining were 80% (95% CI: 0.51–0.95), 75% (95% CI: 0.36–0.96), 85.7% (95% CI: 0.56–0.98), 66.7% (95% CI: 0.31–0.91), and 78.3% (95% CI: 0.58–0.90), respectively.ConclusionsOur study showed that HE staining has relatively moderate accuracy for the diagnosis of HSCR. The use of HE staining is still recommended for the diagnosis of HSCR given the limitation of resource allocation for more expensive IHC technologies in developing countries.

Effects of MTHFR c.677C>T, F2 c.20210G>A and F5 Leiden Polymorphisms in Gastroschisis

ABSTRACT Background: Gastroschisis is a developmental disorder involving the extrusion of fetal intestines through a defect in the abdominal wall. The mechanism is presumed to be a dual vascular/thrombotic pathogenesis, where normal right umbilical vein involution forms a possible site for thrombosis adjacent to the umbilical ring. Purpose: The aim of this study was to demonstrate that the 3 common prothrombotic polymorphisms, MTHFR c.677C>T, F2 c.20210G>A, and F5 Leiden, were elevated in frequency in Indonesian gastroschisis patients. Material and Methods: Three genetic markers were investigated in 46 patients with gastroschisis and 89 ethnicity-matched controls for association studies using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) or TaqMan Genotyping Assays on genomic DNA. Results: MTHFR c.677C>T showed a significant association with gastroschisis (OR = 2.1, 95% CI = 1.13–3.86; p = .018) but no affected infants had risk alleles for either F2 c.20210G>A or F5 Leiden. Further, the frequency of MTHFR risk allele (T) in patients with maternal age <25 years is marginally significant higher than those in cases with maternal age ≥25 years (p = .069) with an OR of 2.7 (95% CI = 0.90–8.07). Conclusions: MTHFR is a common susceptibility factor for gastroschisis in Indonesia. The increased gastroschisis risk in offspring of younger maternal age suggests the thrombotic pathogenesis model. A founder effect is the most likely explanation for the rarity of the F2 and F5 Leiden polymorphisms in Indonesian population.

Novel SCN1A mutations in Indonesian patients with severe myoclonic epilepsy in infancy

Background:u2002 Severe myoclonic epilepsy in infancy (SMEI) and borderline SMEI (SMEB) are caused by a mutation in SCN1A, which encodes a voltage‐gated sodium channel α1‐subunit protein. Although many mutations in SCN1A have been associated with clinical features of SMEI or SMEB from different ethnic groups, there have been no such reports from the South‐East Asian populations so far.

The impact of down-regulated SK3 expressions on Hirschsprung disease

BackgroundSome Hirschsprung’s disease (HSCR) patients showed persistent bowel symptoms following an appropriately performed pull-through procedure. The mechanism is presumed to be down-regulated small-conductance calcium-activated potassium channel 3 (SK3) expression in the HSCR ganglionic intestines. We aimed to investigate the SK3 expression’s impact in HSCR patients after a properly performed pull-through surgery in an Indonesian population, a genetically distinct group within Asia.MethodsWe assessed SK3 gene expression in both the ganglionic and aganglionic colon of HSCR patients and controls colon by quantitative real-time polymerase chain reaction (RT-PCR).ResultsWe ascertained fourteen sporadic HSCR patients and six anorectal malformation patients as controls. Quantitative RT-PCR showed that the SK3 expression was significantly lower (23-fold) in the ganglionic colon group compared to the control group (9.9u2009±u20094.6 vs. 5.4u2009±u20093.4; pu2009=u20090.044). The expression of SK3 in the aganglionic colon group was also significantly lower (43-fold) compared to the control group (10.8u2009±u20094.4 vs. 5.4u2009±u20093.4; pu2009=u20090.015).ConclusionOur study shows that the down-regulated SK3 expression in ganglionic intestines might contribute to the persistent bowel symptoms following a properly performed pull-through surgery in Indonesian HSCR patients. Furthermore, this study is the first report of SK3 expression in a sample population of Asian ancestry.

Hospital-based surveillance of congenital rubella syndrome in Indonesia

AbstractCongenital rubella syndrome (CRS) has serious consequences, such as miscarriage, stillbirth, and severe birth defects in infants, resulting from rubella virus infection during pregnancy. However, rubella vaccine has not yet been implemented in Indonesia. This study aimed (1) to estimate the incidence of CRS in Indonesia, (2) describe the clinical features of CRS at our referral hospital, and (3) pilot a CRS surveillance system to be extended to other hospitals. We conducted a 4-month prospective surveillance study of infants aged <1xa0year with suspected CRS in 2013 at an Indonesian hospital. Infants with suspected CRS were examined for rubella-specific IgM antibody or rubella IgG antibody levels. Of 47 suspected cases of CRS, 11/47 (23.4%), 9/47 (19.1%), and 27/47 (57.5%) were diagnosed as laboratory-confirmed, clinically compatible, and discarded CRS, respectively. The most common defects among laboratory-confirmed CRS cases were hearing impairment (100%), congenital cataracts (72.7%), microcephaly (72.7%), and congenital heart defects (45.5%).n Conclusion: The number of laboratory-confirmed CRS cases among Indonesian infants is high. Furthermore, hearing impairment is the most common clinical feature of CRS in infants. Our findings indicate the importance of implementation of rubella vaccine in Indonesia. Conducting hospital-based surveillance of CRS in other hospitals in Indonesia may be appropriate.What is Known:•Congenital rubella syndrome (CRS) has serious consequences in infants resulting from rubella virus infection during pregnancy.•The incidence of CRS in most developed countries has greatly decreased since implementation of rubella vaccination.•Rubella vaccine has not yet been implemented in many developing countries.What is New:•The number of laboratory-confirmed CRS cases among Indonesian infants was high.•Implementation of rubella vaccine into immunization programs in Indonesia is important because of the high number of CRS cases.•Our study highlights the need for ongoing prospective surveillance of CRS in Indonesia.

Effect of RET c.2307T>G Polymorphism on the Outcomes of Posterior Sagittal Neurectomy for Hirschsprung Disease Procedure in Indonesian Population

We investigated the effect of RET c.2307T>G polymorphism on the outcomes of posterior sagittal neurectomy for Hirschsprung disease (PSNHD) procedure in Indonesia. Hirschsprung disease (HSCR) is a neurocristopathy characterized by absence of enteric ganglia along variable lengths of the intestine in neonates. The RET c.2307T>G polymorphism has been shown to be associated with HSCR. Many surgical techniques with some advantage and disadvantage were established for HSCR. We have conducted PSNHD in short-segment HSCR patients.Thirty-one nonsyndromic HSCR patients underwent PSNHD. The polymorphism was determined using PCR-RFLP in genomic DNA. The rate of enterocolitis and constipation outcomes following PSNHD were 6 (19%) and 4 (13%) patients, respectively. The RET c.2307T>G polymorphism did not influence either enterocolitis or constipation outcome following PSNHD at P value of 0.07 (OR = 0.28; 95% CI = 0.08-1.05) and 0.67 (OR = 0.58; 95% CI = 0.12-2.76), respectively. Our study suggested that RET c.2307T>G polymorphism may not affect outcomes of PSNHD procedure in Indonesia. Furthermore, a multicenter study with a larger sample size is necessary to clarify this result.