Aberrant SOX10 and RET expressions in patients with Hirschsprung disease.

BACKGROUND: HSCR is a complex genetic disorder characterized by the absence of ganglion cells in the intestine, leading to a functional obstruction. It is due to a disruption of complex signaling pathways within the gene regulatory network (GRN) during the development of the enteric nervous system (ENS), including SRY-Box Transcription Factor 10 (SOX10) and REarranged during Transfection (RET). This study evaluated the expressions of SOX10 and RET in HSCR patients in Indonesia. METHODS: Total RNA of 19 HSCR ganglionic and aganglionic colons and 16 control colons were analyzed using quantitative real-time polymerase chain reaction for SOX10 and RET with GAPDH as the reference gene. Livak's method (2-ΔΔCT) was used to determine the expression levels of SOX10 and RET. RESULTS: Most patients were males (68.4%), in the short aganglionosis segment (78.9%), and had undergone transanal endorectal pull-through (36.6%). There were significant upregulated SOX10 expressions in both ganglionic (2.84-fold) and aganglionic (3.72-fold) colon of HSCR patients compared to controls' colon (ΔCT 5.21 ± 2.04 vs. 6.71 ± 1.90; p = 0.032; and ΔCT 4.82 ± 1.59 vs. 6.71 ± 1.90; p = 0.003; respectively). Interestingly, the RET expressions were significantly downregulated in both ganglionic (11.71-fold) and aganglionic (29.96-fold) colon of HSCR patients compared to controls' colon (ΔCT 12.54 ± 2.21 vs. 8.99 ± 3.13; p = 0.0004; and ΔCT 13.90 ± 2.64 vs. 8.99 ± 3.13; p = 0.0001; respectively). CONCLUSIONS: Our study shows aberrant SOX10 and RET expressions in HSCR patients, implying the critical role of SOX10 and RET in the pathogenesis of HSCR, particularly in the Indonesian population. Our study further confirms the involvement of SOX10-RET within the GNR during the ENS development.

Sequence analysis of the Spike, RNA-dependent RNA polymerase, and protease genes reveals a distinct evolutionary pattern of SARS-CoV-2 variants circulating in Yogyakarta and Central Java provinces, Indonesia.

During the Covid-19 pandemic, the resurgence of SARS-CoV-2 was due to the development of novel variants of concern (VOC). Thus, genomic surveillance is essential to monitor continuing evolution of SARS-CoV-2 and to track the emergence of novel variants. In this study, we performed phylogenetic, mutation, and selection pressure analyses of the Spike, nsp12, nsp3, and nsp5 genes of SARS-CoV-2 isolates circulating in Yogyakarta and Central Java provinces, Indonesia from May 2021 to February 2022. Various bioinformatics tools were employed to investigate the evolutionary dynamics of distinct SARS-CoV-2 isolates. During the study period, 213 and 139 isolates of Omicron and Delta variants were identified, respectively. Particularly in the Spike gene, mutations were significantly more abundant in Omicron than in Delta variants. Consistently, in all of four genes studied, the substitution rates of Omicron were higher than that of Delta variants, especially in the Spike and nsp12 genes. In addition, selective pressure analysis revealed several sites that were positively selected in particular genes, implying that these sites were functionally essential for virus evolution. In conclusion, our study demonstrated a distinct evolutionary pattern of SARS-CoV-2 variants circulating in Yogyakarta and Central Java provinces, Indonesia.

Collagen gene cluster expression and liver fibrogenesis in patients with biliary atresia: a preliminary study.

OBJECTIVE: Biliary atresia (BA) is a progressive fibro-obliterative disease of the biliary tract, which results in end-stage liver disease. However, liver fibrosis progression may continue even after Kasai surgery. Recent evidence showed that collagen plays a pivotal role in the progression of liver fibrosis in BA. However, most studies were conducted in developed countries. We investigated the expressions of the collagen gene cluster (COL6A1, COL6A2, COL6A3, and COL1A1) in BA patients in Indonesia. RESULTS: There was a significant down-regulated expression of COL6A1 (ΔCT 9.06 ± 2.64 vs. 5.42 ± 2.41; p = 0.0009), COL6A2 (ΔCT 8.25 ± 2.07 vs. 5.77 ± 3.51; p = 0.02), COL6A3 (ΔCT 11.2 ± 6.08 vs. 6.78 ± 3.51; p = 0.024), and COL1A1 (ΔCT 3.26 ± 1.71 vs. 0.19 ± 2.76; p = 0.0015) in BA patients compared to controls. Interestingly, the collagen gene cluster expressions were significantly associated with the presence of cirrhosis (p = 0.0085, 0.04, and 0.0283 for COL6A1, COL6A2, and COL6A3, respectively). In conclusion, our study shows the changes in the collagen gene cluster, particularly collagen type I and VI, expressions in patients with BA in a particular developing country. Our findings suggest the role of these collagen gene clusters in the liver fibrogenesis of BA.

Identification of the genetic basis of pediatric neurogenetic disorders at a tertiary referral hospital in Indonesia: Contribution of whole exome sequencing.

BACKGROUND: Neurogenetic disorders (NGDs) are complex Mendelian disorders that affect the neurological system. A molecular diagnosis will provide more information about pathophysiology, prognosis, and therapy, including future genetic therapy options. Whole-Exome Sequencing (WES) can rapidly discover the genetic basis in NGDs. OBJECTIVE: The purpose of this study was to assess the WES results and its value in diagnosing pediatric NGDs, especially those with unspecified clinical features. METHODS: A retrospective chart review was performed from May 2021- February 2023 in Dr. Sardjito General Hospital, a tertiary referral hospital in Yogyakarta, Indonesia. WES proband only was conducted on children aged 0 to 17 years old who met one or more of the following criteria: (1) epileptic encephalopathy and familial epilepsy; (2) complex neurodevelopmental phenotypes; (3) leukodystrophy; (4) movement disorders; and (5) neurocutaneous disorder. The WES was conducted in the certified laboratory, 3Billion, in Seoul, Korea. RESULTS: The diagnosis yield of WES in our study was 45% (9/20). We identified nine positive results, including eight pathogenic single nucleotide variants (SNVs) in 8 genes (KCNQ2, ARSA, UBE3A, IRF2BPL, ATM, MECP2, TSC2, and NF1), and one variant with uncertain significance (VUS) in the ADK gene that has not been able to explain the observed clinical features. Of the nine patients with positive WES results, five had missense mutations, three frameshift mutations, and one nonsense mutation. Additionally, we identified two suggestive copy number variants (CNVs) in 15q11.2q13.1 and 1p31.3. CONCLUSIONS: Whole-Exome Sequencing is an essential diagnostic tool for pediatric NGDs, especially those with unspecified clinical features. It ends multi-year diagnostic odysseys, provides personalized medicine therapy, and optimizes genetic counselling for these families.

Prognostic factors for the outcomes of COVID-19 patients infected with SARS-CoV-2 Omicron and Delta variants.

BACKGROUND: The SARS-CoV-2 Omicron variant has replaced the previously dominant Delta variant because of high transmissibility. However, studies on the impact of the Omicron variant on the severity of COVID-19 are still limited in developing countries. Our study aimed to determine the prognostic factors for the outcomes of patients infected with SARS-CoV-2 Omicron and Delta variants, including age, sex, comorbidities, and smoking. METHODS: In this retrospective cross-sectional study, we involved 352 patients with COVID-19 from Yogyakarta and Central Java provinces, Indonesia, from May 2021 to February 2022, consisting of 164 males and 188 females. We included all patients with the PCR's Ct value of less than 30 for further whole-genome sequencing. RESULTS: Ct value and mean age of COVID-19 patients were not significantly different between both groups (p = 0.146 and 0.273, respectively). Patients infected with Omicron (n = 139) and Delta (n = 213) variants showed similar hospitalization (p = 0.396) and mortality rates (p = 0.565). Multivariate analysis of both groups showed that older age (≥ 65 years) had a higher risk for hospitalization (OR = 3.86 [95% CI = 1.29-11.5]; p = 0.015) and fatalities (OR = 3.91 [95% CI = 1.35-11.42]; p = 0.012). In both groups, patients with cardiovascular disease had a higher risk for hospitalization (OR = 5.36 [95% CI = 1.08-26.52]; p = 0.039), whereas patients with diabetes revealed a higher risk for fatalities (OR = 9.47 [95% CI = 3.23-27.01]; p = < 0.001). CONCLUSIONS: Our study shows that patients infected with Omicron and Delta variants reveal similar clinical outcomes, including hospitalization and mortality. Our findings further confirm that older age, cardiovascular disease, and diabetes are substantial prognostic factors for the outcomes of COVID-19 patients. Our findings imply that COVID-19 patients with older age, cardiovascular disease, or diabetes should be treated comprehensively and cautiously to prevent further morbidity and mortality. Furthermore, incomplete data on vaccination status hampered us from analyzing further its impact on hospitalization and mortality in our patients.

De novo mutation of the TSC2 gene in patient with Tuberous Sclerosis Complex-Associated Neuropsychiatric Disorders (TAND) Phenotype: a case report.

Tuberous sclerosis complex (TSC) is a neurocutaneous disease caused by a mutation in the TSC1 or TSC2 gene. There are several neuropsychiatric manifestations associated with TSC known as TSC-associated neuropsychiatric disorder (TAND). This article concerns neuropsychiatric manifestations in children with the TSC2 gene mutation, with genetic analysis findings using whole-exome sequencing. Case: A 17-year-old girl presented with TSC, absence and focal epilepsy, borderline intellectual functioning, organic psychosis, and renal angiomyolipoma. She was emotionally unstable and preoccupied with irrelevant fears. In the physical examination, we found multiple hypomelanotic maculae, angiofibroma, and a shagreen patch. The intellectual assessment result with the Wechsler Adult Intelligence Scale at 17 was borderline intellectual functioning. Brain MRI showed cortical and subcortical tubers in the parietal and occipital lobes. Whole-exome sequencing was conducted, and the result was a missense mutation in exon 39 of the TSC2 gene [NM_000548.5:c.5024C>T (NP_000539.2:p.Pro1675Leu)]. The Sanger sequencing of the patient's parents revealed no mutations in the TSC2 gene, confirming the patient's de novo mutation. The patient was given several antiepileptic and antipsychotic drugs. Clinical discussion: Neuropsychiatric manifestation is a common phenotype in the TSC variant, and psychosis is one of the rare TAND symptoms in children. Conclusions: The neuropsychiatric phenotype and genotype in TSC patients are rarely reported and evaluated. We reported a female child with epilepsy, borderline intellectual functioning, and organic psychosis associated with a de novo mutation of the TSC2 gene. Organic psychosis is a rare symptom of TAND which also manifested in our patient.

Phenotypes of a female patient with novel de novo frameshift ARX variant identified by whole-exome sequencing: a case report.

Variants in the aristaless-related homeobox (ARX) gene cause a diverse spectrum of phenotypes of neurodevelopmental disorders (NDD) in male patients. This article describes the role of genetic testing using whole-exome sequencing (WES) in detecting a novel de novo frameshift variant in the ARX gene in a female patient with autism, seizure, and global developmental delay. Case presentation: A 2-year-old girl with frequent seizures, global developmental delay, and autistic features was referred to our hospital. She was the second child of consanguineous non-affected parents. She had a high forehead, mildly prominent ears, and prominent nasal root. A generalized epileptiform discharge was noted in her electroencephalography. Brain MRI revealed corpus callosum agenesis, cerebral atrophy, and a left parafalcine cyst. The WES result showed a likely pathogenic variant identified as a novel de novo deletion in exon 4 of the ARX gene, which creates a frameshift variant. The patient is on dual therapy of antiepilepsy drugs, physiotherapy, speech therapy, occupational therapy, and oral motor exercises. Clinical discussion: Variants in the ARX gene can result in various phenotypes in males transmitted from asymptomatic carrier females. However, several reports showed that the ARX variants might cause phenotypes in females with milder symptoms than affected males. Conclusion: We report a novel de novo ARX variant in an affected female with a NDD. Our study confirms that the ARX variant might cause remarkable pleiotropy phenotypes in females. Moreover, WES could help to identify the pathogenic variant in NDD patients with diverse phenotypes.

Neurological Manifestations of COVID-19 Associated Multisystem Inflammatory Syndrome in Children (MIS-C) in Yogyakarta, Indonesia.

OBJECTIVE: This observational cohort study aims to provide data on pediatric patients with neurological manifestations associated with multisystem inflammatory syndrome in children (MIS-C) associated with coronavirus disease (Covid-19). METHODS: Patients aged <18 with neurologic symptoms and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection from January, 2021 to January, 2022 at the Dr. Sardjito Hospital in Yogyakarta, Indonesia were evaluated. We used WHO diagnostic criteria to classify patients as MIS-C or non-MIS-C. Demographic information, symptoms, and outcomes were compared between MIS-C and non-MIS-C groups. RESULTS: Between January, 2021 and January, 2022, 74 pediatric patients were considered eligible. More than half of the patients were female (54.1%), and 24.3% presented with MIS-C. Length of hospitalization was significantly longer in MIS-C individuals (P=0.006). The commonest neurological findings were involuntary movements (43.2%) and paresis (27%). The commonest neuroimaging findings were meningoencephalitis (18.9%) and hydrocephalus (22.9%). Among all the variety of neurologic manifestations in non-MIS-C and MIS-C patients, a statistically significant result was found for fever (71.4% vs 100%; P=0.015), altered mental state (14.2% vs 50%, P=0.004), and paresis (33.9% vs 5.5%, P=0.030). CONCLUSION: MIS-C was found in 24% of our patients with acute neurologic symptoms, and most cases (51.8%) had positive SARS-CoV-2 antibody results.

The importance of prenatal diagnosis for the surgical strategy of giant cystic meconium peritonitis: A case report.

Background: Giant cystic meconium peritonitis (MP) is a relatively rare entity. Prompt surgical treatment is required to manage the underlying etiology and reestablish the continuity of the intestines. Despite perinatal and postoperative care improvements, the overall mortality rate is still relatively high. We reported a giant cystic MP that was recognized using antenatal sonography (US). It was successfully treated with primary anastomosis. Case presentation: We presented a female newborn with a chief complaint of abdominal mass. The prenatal sonography showed an intraabdominal cyst at the 28th week of gestation. She was born at the gestational age of 38 weeks via vaginal delivery from a primigravid mother without complications, with a birth weight of 3275 g. Elective surgery was performed at the age of eight days, and a calcified 10 cm cyst was revealed along with severe adhesions. The cyst was found to communicate with the ileum located 30 cm proximal from the ileocecal junction. No malrotation and volvulus were found. The cyst and a portion of the ileum were resected, followed by a primary end-to-end anastomosis. Pathologic examination showed necrotic tissue lined with epithelial tissue with microcalcifications containing bilirubin pigments, consistent with cystic MP. The patient has uneventfully discharged on postoperative day 17. The patient has normal growth and development, except for delayed walking, at the last follow-up of two years of age. Conclusion: Giant cystic MP is a rare disorder that can be detected early using the antenatal US. Our case highlights the importance of early diagnosis for giant cystic MP using the antenatal US leads to prompt surgical treatment and a more favorable prognosis.

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.

Phylogenetic analysis of congenital rubella virus from Indonesia: a case report.

BACKGROUND: Rubella is a common inherited infection resulting in congenital cataracts and a significant cause of permanent vision loss in developing countries. In 2016, Indonesia had the highest number of congenital rubella syndrome (CRS) cases globally. Here, we report the first genotype of the rubella virus extracted from the eye lens from a child with congenital cataracts due to CRS. CASE PRESENTATION: A female neonate was delivered by an elective caesarean delivery with normal birth weight at term from a 24-year-old mother in the rural setting. The baby presented with bilateral congenital cataracts, small-moderate secundum atrial septal defect, severe supravalvular pulmonary stenosis, and profound bilateral hearing loss. She also had microcephaly and splenomegaly. The patient's serology showed persistent positive IgG for rubella virus at the age of four years and four months. Following extraction during cataract surgery, viral detection of the lenses identified the presence of rubella. Phylogenetic analysis confirmed that the virus was grouped into genotype 1E. CONCLUSIONS: Our study reports the first phylogenetic analysis of the rubella virus extracted from the eye lens of a child with CRS in Indonesia. The detection of the rubella virus from eye lenses is remarkably promising. Our findings also emphasize the importance of molecular epidemiology in tracking the origin of rubella infection toward achieving virus eradication.

Autosomal dominant Emery-Dreifuss muscular dystrophy caused by a mutation in the lamin A/C gene identified by exome sequencing: a case report.

BACKGROUND: Emery-Dreifuss Muscular Dystrophy (EDMD) is an uncommon genetic disease among the group of muscular dystrophies. EDMD is clinically heterogeneous and resembles other muscular dystrophies. Mutation of the lamin A/C (LMNA) gene, which causes EDMD, also causes many other diseases. There is inter and intrafamilial variability in clinical presentations. Precise diagnosis can help in patient surveillance, especially before they present with cardiac problems. Hence, this paper shows how a molecular work-out by next-generation sequencing can help this group of disorders. CASE PRESENTATION: A 2-year-10-month-old Javanese boy presented to our clinic with weakness in lower limbs and difficulty climbing stairs. The clinical features of the boy were Gower's sign, waddling gait and high CK level. His father presented with elbow contractures and heels, toe walking and weakness of limbs, pelvic, and peroneus muscles. Exome sequencing on this patient detected a pathogenic variant in the LMNA gene (NM_170707: c.C1357T: NP_733821: p.Arg453Trp) that has been reported to cause Autosomal Dominant Emery-Dreifuss muscular dystrophy. Further examination showed total atrioventricular block and atrial fibrillation in the father. CONCLUSION: EDMD is a rare disabling muscular disease that poses a diagnostic challenge. Family history work-up and thorough neuromuscular physical examinations are needed. Early diagnosis is essential to recognize orthopaedic and cardiac complications, improving the clinical management and prognosis of the disease. Exome sequencing could successfully determine pathogenic variants to provide a conclusive diagnosis.

Dp71 and intellectual disability in Indonesian patients with Duchenne muscular dystrophy.

INTRODUCTIONS: Duchenne muscular dystrophy (DMD) is an X-linked recessive progressive muscular disease marked by developmental delays due to mutations in the DMD gene, which encodes dystrophin. Brain comorbidity adds to the burden of limited mobility and significantly impacts patients' quality of life and their family. The changes of expression of dystrophin isoforms in the brain due to DMD gene mutations are thought to be related to the cognitive and neurobehavior profiles of DMD. OBJECTIVES: This cross-sectional study aimed to characterize cognitive and neurodevelopmental profiles of patients with DMD and to explore underlying genotype-phenotype associations. METHODS: Patients with DMD aged 5-18 years from Dr Sardjito Hospital and Universitas Gadjah Mada Academic Hospital from 2017-2022 were included. Multiplex ligation-dependent probe amplification and whole exome sequencing were used to determine mutations in the DMD genes. Cognitive function was measured by intelligence quotient testing using the Wechsler Intelligence Scale for Children and adaptive function tests with Vineland Adaptive Behavior Scales. The Autism Mental Status Exam and Abbreviated Conner's Rating Scale were used to screen for autism spectrum disorder (ASD) and attention deficit and hyperactivity disorder (ADHD), respectively. RESULTS: The mean total IQ score of DMD patients was lower than that of the general population (80.6 ± 22.0 vs 100 ± 15), with intellectual disability observed in 15 boys (29.4%). Of the 51 patients with DMD, the Dp71 group had the lowest cognitive performance with a total IQ score (46 ± 24.8; p = 0.003), while the Dp427 group and Dp140 group's total IQ scores were 83.0 ± 24.6 and 84.2 ± 17.5 respectively. There were no DMD patients with ASD, while 4 boys (7.8%) had comorbidity with ADHD. CONCLUSION: Boys with DMD are at higher risk of intellectual disability. The risk appears to increase with mutations at the 3' end of the gene (Dp71 disruption). Moreover, Dp71 disruption might not be associated with ADHD and ASD in patients with DMD.

Association between prognostic factors and the clinical deterioration of preterm neonates with necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is responsible for most morbidity and mortality in neonates. Early recognition of the clinical deterioration in newborns with NEC is essential to enhance the referral and management and potentially improve the outcomes. Here, we aimed to identify the prognostic factors and associate them with the clinical deterioration of preterm neonates with NEC. We analyzed the medical records of neonates with NEC admitted to our hospital from 2016 to 2021. We ascertained 214 neonates with NEC. The area under the receiver operating characteristic (ROC) curve and cut-off level of age at onset, C-reactive protein (CRP), leukocyte count, and platelet count for the clinical deterioration of preterm neonates with NEC was 0.644 and 10.5 days old, 0.694 and 4.5 mg/L, 0.513 and 12,200/mm3, and 0.418 and 79,500/mm3, respectively. Late-onset, history of blood transfusion, thrombocytopenia, and elevated CRP were significantly associated with the clinical deterioration of neonates with NEC (p = < 0.001, 0.017, 0.001, and < 0.001, respectively), while leukocytosis, gestational age, and birth weight were not (p = 0.073, 0.274, and 0.637, respectively). Multivariate analysis revealed that late-onset and elevated CRP were strongly associated with the clinical deterioration of neonates with NEC, with an odds ratio of 3.25 (95% CI = 1.49-7.09; p = 0.003) and 3.53 (95% CI = 1.57-7.95; p = 0.002), respectively. We reveal that late-onset and elevated CRP are the independent prognostic factor for the clinical deterioration of preterm neonates with NEC. Our findings suggest that we should closely monitor preterm neonates with NEC, particularly those with late-onset of the disease and those with an elevated CRP, to prevent further clinical deterioration and intervene earlier if necessary.

Long-term functional outcomes of patients with Hirschsprung disease following pull-through.

BACKGROUND: Hirschsprung disease (HSCR) is a common congenital disorder presenting with functional obstruction due to aganglionosis of the colon. There are numerous types of pull-through surgery for managing HSCR, such as transabdominal endorectal (Soave), Swenson, Duhamel, transanal endorectal pull-through (TEPT), and laparoscopic (Georgeson) approach. Here, we aimed to describe the long-term outcome of patients with HSCR who underwent transabdominal Soave, Duhamel, and TEPT in our institution. METHODS: We performed a cross-sectional analysis for patients who underwent Duhamel, Soave, and TEPT at our institution from January 2012 to December 2015. Long-term functional outcome was determined by bowel function score (BFS). The BFS was obtained by interviewing patients who had completed at least three years of follow-up. RESULTS: Twenty-five patients were included in this study who underwent transabdominal Soave (n = 8), Duhamel (n = 4), and TEPT (n = 13). There were 24 patients with short aganglionosis type. The median age of HSCR diagnosis was 10 (IQR = 1-39) months, while the median age of pull-through surgery was 17 (IQR = 7-47) months. The median follow-up of BFS level for HSCR patients after pull-through was 72 (IQR, 54-99) months. There were 11 patients with good BFS level and 10 patients with normal BFS level. Additionally, 50% of Duhamel patients had poor BFS level, while 50% of Soave patients had good BFS level, and 54% of TEPT patients had normal BFS level (p = 0.027). As many as 50% of Duhamel patients showed daily soiling and required protective aids, while 38.5% of TEPT had staining less than 1/week and no change of underwear required, and 50% of Soave patients revealed no soiling, respectively (p = 0.030). Furthermore, 75% of Duhamel patients had accidents, while 75% of Soave and 46.2% of TEPT patients had no accidents (p = 0.035). CONCLUSION: Our study shows that the type of definitive surgery might affect the long-term bowel functional outcome; particularly, the TEPT approach might have some advantages over the transabdominal Soave and Duhamel procedures.

Is There Any Mosaicism in REarranged During Transfection Variant in Hirschsprung Disease's Patients?

Background: Hirschsprung disease (HSCR) is a heterogeneous genetic disease characterized by the absence of ganglion cells in the intestinal tract. The REarranged during Transfection (RET) is the most responsible gene for its pathogenesis. RET's somatic mosaicisms have been reported for HSCR; however, they are still under-recognized. Therefore, we determined the frequency of somatic mutation of RET rs2435357 in HSCR patients at our institution. Methods: We performed RET rs2435357 genotyping from 73 HSCR formalin-fixed and paraffin-embedded (FFPE) rectal and 60 non-HSCR controls using the PCR-RFLP method. Subsequently, we compared those frequencies of genotypes for RET rs2435357 with our previous genotyping data from 93 HSCR blood specimens. Results: The frequencies of genotypes for RET rs2435357 in HSCR paraffin-embedded rectal were CC 0, CT 11 (15%), and TT 62 (85%), whereas their frequencies in HSCR blood samples were CC 4 (4.3%), CT 22 (23.7%), and TT 67 (72%). Those frequencies differences almost reached a significant level (p = 0.06). Moreover, the frequency of RET rs2435357 risk allele (T) was significantly higher in HSCR patients (135/146, 92.5%) than controls (46/120, 38.3%) (p = 3.4 × 10-22), with an odds ratio of 19.74 (95% confidence interval = 9.65-40.41). Conclusion: Our study suggests somatic mosaicism in HSCR patients. These findings further imply the complexity of the pathogenesis of HSCR. Moreover, our study confirms the RET rs2435357 as a significant genetic risk factor for HSCR patients.

The impact of NRG1 expressions and methylation on multifactorial Hirschsprung disease.

BACKGROUND: Hirschsprung disease (HSCR) is a complex genetic disorder characterized by the lack of ganglion cells in the intestines. A current study showed that the NRG1 rare variant frequency in Indonesian patients with HSCR is only 0.9%. Here, we investigated the impact of NRG1 expressions and methylation patterns on the pathogenesis of HSCR. METHODS: This cross-sectional study determined NRG1 type I (HRGα, HRGß1, HRGß2, HRGß3, HRGγ, and NDF43 isoforms), type II and type III expressions in both ganglionic and aganglionic colons of 20 patients with HSCR and 10 control colons by real-time polymerase chain reaction (qPCR). For methylation studies, we treated the extracted gDNA from 16 HSCR patients' and 17 control colons with sodium bisulfate and analyzed the methylation pattern of NRG1 exon 1 with methylation-specific PCR. The samples were collected and analyzed at our institution from December 2018 to December 2020. RESULTS: NRG1 types I, II and III expressions were upregulated (17.2-, 3.2-, and 7.2-fold, respectively) in the ganglionic colons compared with control colons (type I: 13.32 ± 1.65 vs. 17.42 ± 1.51, p < 0.01; type II: 13.73 ± 2.02 vs. 16.29 ± 2.19, p < 0.01; type III: 13.47 ± 3.01 vs. 16.32 ± 2.58, p = 0.03; respectively); while only type I (7.7-fold) and HRGß1/HRGß2 (3.3-fold) isoforms were significantly upregulated in the aganglionic colons compared to the controls (type I: 14.47 ± 1.66 vs. 17.42 ± 1.51, p < 0.01; HRGß1/HRGß2: 13.62 ± 3.42 vs 14.75 ± 1.26, p = 0.01). Moreover, the frequency of partially methylated NRG1 was higher in the ganglionic (81%) and aganglionic (75%) colons than in the controls (59%). CONCLUSIONS: Our study provides further insights into the aberrant NRG1 expression in the colons of patients with HSCR, both ganglionic and aganglionic bowel, which might contribute to the development of HSCR, particularly in Indonesia. Furthermore, these aberrant NRG1 expressions might be associated with its methylation pattern.

The role of whole exome sequencing in the UBE3A point mutation of Angelman Syndrome: A case report.

INTRODUCTION: Angelman Syndrome (AS) is a rare disorder with a relatively well-defined phenotype caused by lack of expression of the maternally inherited ubiquitin-protein ligase E3A (UBE3A) gene in the brain. This article describes the role of genetic testing using whole-exome sequencing (WES) in detecting rare AS variants, a point mutation in the UBE3A gene. CASE PRESENTATION: We describe a rarely reported clinical presentation of AS in a two year and ten months old girl with severe developmental delay, movement and balance disorder, frequent smiling, apparent happy demeanor, speech impairment, absence of seizure, lack of sleep, and abnormal food-related behavior. Physical examination showed microcephaly, with facial characteristics of AS, ataxia gait, and truncal hypotonia. The electroencephalogram showed medium amplitude rhythmic 2-3c/s. Brain Magnetic Resonance Imaging revealed microcephaly, corpus callosum dysgenesis, and heterotopia grey matter on the bilateral lateral ventricle. WES was conducted to search pathogenic variants and showed a heterozygous mutation in exon 9 of the UBE3A gene, c.1513C > T (p.Arg505Ter). CONCLUSION: Angelman syndrome is a neurodevelopmental disorder that has several underlying genetic etiologies. WES could detect a rare variant of Angelman syndrome, identified as the point mutation of the UBE3A gene, which cannot be seen with other modalities.

Mutation spectrum analysis of DMD gene in Indonesian Duchenne and Becker muscular dystrophy patients.

Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the DMD gene. The full mutation spectrum of the DMD gene in Indonesian patients is currently unknown. Mutation-specific therapies are currently being developed, such as exon skipping or stop codon read-through therapy. This study was conducted with the aim of identifying the mutation spectrum of the DMD gene in Indonesia to guide future development and application of feasible therapeutic strategies. Methods: This study is a cross sectional study that enrolled 43 male patients with a clinical suspicion of DMD or BMD. Multiplex ligation-dependent probe amplification (MLPA) reaction was performed to screen for the common mutations in the DMD gene. Results: Out of 43 subjects, deletions accounted for 69.77% (n=30) cases, while duplications were found in 11.63% (n=5) cases. One novel duplication spanning exons 2 to 62 was identified. Deletion mutations clustered around the distal (66.67%) and proximal (26.67%) hot spot regions of the DMD gene while duplication mutations were observed solely at the proximal region. Two false positive cases of single exon deletion detected through MLPA were attributed to sequence mutations affecting primer ligation sites, confirming the need to validate all single exon deletions when using this screening method. Analysis of available maternal DNA samples showed that the rate of de novo mutations (48.15%) appears higher than expected in this population. Out of 31 patients who were classified as DMD based on clinical and genotype characterizations, 60.47% (n=26) of cases were suitable for exon skipping therapy. Conclusion: This is the first comprehensive study showing the feasibility of implementing the MLPA method for routine screening of DMD patients in Indonesia. This is also the first study showing the potential applicability of exon skipping therapy in the majority of DMD cases in the country.

Is the Infection of the SARS-CoV-2 Delta Variant Associated With the Outcomes of COVID-19 Patients?

Background: Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) Delta variant (B.1.617.2) has been responsible for the current increase in Coronavirus disease 2019 (COVID-19) infectivity rate worldwide. We compared the impact of the Delta variant and non-Delta variant on the COVID-19 outcomes in patients from Yogyakarta and Central Java provinces, Indonesia. Methods: In this cross-sectional study, we ascertained 161 patients, 69 with the Delta variant and 92 with the non-Delta variant. The Illumina MiSeq next-generation sequencer was used to perform the whole-genome sequences of SARS-CoV-2. Results: The mean age of patients with the Delta variant and the non-Delta variant was 27.3 ± 20.0 and 43.0 ± 20.9 (p = 3 × 10-6). The patients with Delta variant consisted of 23 males and 46 females, while the patients with the non-Delta variant involved 56 males and 36 females (p = 0.001). The Ct value of the Delta variant (18.4 ± 2.9) was significantly lower than that of the non-Delta variant (19.5 ± 3.8) (p = 0.043). There was no significant difference in the hospitalization and mortality of patients with Delta and non-Delta variants (p = 0.80 and 0.29, respectively). None of the prognostic factors were associated with the hospitalization, except diabetes with an OR of 3.6 (95% CI = 1.02-12.5; p = 0.036). Moreover, the patients with the following factors have been associated with higher mortality rate than the patients without the factors: age ≥65 years, obesity, diabetes, hypertension, and cardiovascular disease with the OR of 11 (95% CI = 3.4-36; p = 8 × 10-5), 27 (95% CI = 6.1-118; p = 1 × 10-5), 15.6 (95% CI = 5.3-46; p = 6 × 10-7), 12 (95% CI = 4-35.3; p = 1.2 × 10-5), and 6.8 (95% CI = 2.1-22.1; p = 0.003), respectively. Multivariate analysis showed that age ≥65 years, obesity, diabetes, and hypertension were the strong prognostic factors for the mortality of COVID-19 patients with the OR of 3.6 (95% CI = 0.58-21.9; p = 0.028), 16.6 (95% CI = 2.5-107.1; p = 0.003), 5.5 (95% CI = 1.3-23.7; p = 0.021), and 5.8 (95% CI = 1.02-32.8; p = 0.047), respectively. Conclusions: We show that the patients infected by the SARS-CoV-2 Delta variant have a lower Ct value than the patients infected by the non-Delta variant, implying that the Delta variant has a higher viral load, which might cause a more transmissible virus among humans. However, the Delta variant does not affect the COVID-19 outcomes in our patients. Our study also confirms that older age and comorbidity increase the mortality rate of patients with COVID-19.