A systematic review on Treacher Collins syndrome: Correlation between molecular genetic findings and clinical severity.

Treacher Collins syndrome (TCS, OMIM: 154500) is a rare congenital craniofacial disorder that is caused by variants in the genes TCOF1, POLR1D, POLR1C, and POLR1B. Studies on the association between phenotypic variability and their relative variants are very limited. This systematic review summarized the 53 literatures from PubMed and Scopus to explore the potential TCS genotype-phenotype correlations with statistical analysis. Studies reporting both complete molecular genetics and clinical data were included. We identified that the molecular anomaly within TCOF1 (88.71%) accounted for most TCS cases. The only true hot spot for TCOF1 was detected in exon 24, with recurrent c.4369_4373delAAGAA variant is identified. While the hot spot for POLR1D, POLR1C, and POLR1B were identified in exons 3, 8, and 15, respectively. Our result suggested that the higher severity level was likely to be observed in Asian patients harboring TCOF1 variants rather than POLR1. Moreover, common 5-bp deletions tended to have a higher severity degree in comparison to any variants within exon 24 of TCOF1. In summary, this report suggested the relationship between genetic and clinical data in TCS. Our findings could be used as a reference for clinical diagnosis and further biological studies.

Comparative performance of ELISA and dot blot assay for TSH-receptor antibody detection in Graves' disease.

BACKGROUND: Graves' disease (GD) is an autoimmune disease, and it accounts for major cases of hyperthyroidism. Antibody against thyroid-stimulating hormone receptor/TSHR (TRAb) is responsible for hyperthyroidism and is considered as a diagnostic marker for GD. Therefore, we developed a recombinant protein of human TSHR-169 (hTSHR-169), which was specifically recognized TRAb in the serum of GD patients and then compare the diagnostic performance between ELISA and dot blot of TRAb tests for their ability to diagnose GD. METHODS: 20 GD patients and 20 healthy individuals from the Indonesian population were enrolled. TRAb concentration and density were quantified. Comparative analysis was performed using receiver-operating curve (ROC) analysis. RESULTS: For dot blot assay, the minimum concentration to detect TRAb requiring 100 ng of antigen with antiserum diluted at 1:60. For diagnosing GD, the ELISA yielded a higher AUC compared with the dot blot assay (0.95 and 0.85, respectively). Using the recommended cutoff values, the efficiency of both assays was examined by comparing the specificity and sensitivity of the assays to the clinical diagnosis. The ELISA showed 80% and 95%, while the dot blot assay showed 70% and 95% sensitivity and specificity, respectively. CONCLUSION: Although the dot blot assay exhibited lower performance than the ELISA method, the dot blot assay is a simple and rapid diagnostic assay that is suitable for diagnosing GD in rural areas, in which healthcare facilities sometimes are not accessible.

Cloning and Protein Expression of eccB5 Gene in ESX-5 System from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis in human. One of the major M. tuberculosis virulence factors is early secretory antigenic target of 6-kDa (ESAT-6), and EccB5 protein encoded by eccB5 is one of its components. EccB5 protein is a transmembrane protein in ESX-5 system. The aim of this study is to explore the characteristics of wild-type EccB5 and its mutant form N426I. We expressed the EccB5 protein by cloning the mutant and wild-type eccB5 gene in Escherichia coli (E. coli). We compared the protein structure of wild type and mutant form of EccB5 and found changes in structure around Asn426 (loop structure) in wild type and around Ile426 (ß-strand) in the mutant. The truncated recombinant protein of EccB5 was successfully cloned and expressed using plasmid pCold I in E. coli DH5α and E. coli strain Rosetta-gami B (DE3) and purified as a 38.6 kDa protein by using the affinity column. There was no detectable adenosine triphosphatase activity in truncated forms of EccB5 and its mutant. In conclusion, our study reveals successful cloning and protein expression of truncated form of eccB5 gene of M. tuberculosis. EccB5 protein in ESX-5 system may be an important membrane component involved in the transport machinery of type VII secretion system, which is essential for growth and virulence.

SINGLE NUCLEOTIDE POLYMORPHISM OF ECCB5 GENE OF MYCOBACTERIUM TUBERCULOSIS COMPLEX ISOLATES FROM SUSPECTED PULMONARY TB PATIENTS IN SURABAYA INDONESIA.

BACKGROUND: Mycobacterium tuberculosis Complex (MTBC) is a group of Mycobacterium that causes tuberculosis (TB). TB is an infectious disease that remains a global health problem. Indonesia is one of the five countries in the world where TB is the most prevalent and became the country with tle second largest rate of TB in 2014 and 2015. MTBC has high pathogenicity that can cause infections in animals and humans. The most common route of transmission is via airborne droplet nuclei and contact with animals or humans infected with TB. MTBC has many virulence factors. One of these factors is EccB5 that is encoded by eccB5 gene. EccB5 is a transmembrane protein-conserved membrane protein and could play a role in inducing damage in host cells, macrophage infection, and may correlate with active disease. The characterization of eccB5 gene needs to be studied to determine the nucleotide sequences, which may be associated with active disease. The aim of this research was to analyze the nuclotide sequences of eccB5 gene of MTBC from suspected pulmonary tuberculosis patients, SNPs of eccB5 gene and possible correlation with the disease, especially in Indonesia. MATERIALS AND METHODS: Samples were collected from the Tuberculosis Laboratory, Clinical Microbiology of Dr. Soetomo Hospital Surabaya Indonesia. DNA extraction used boiling extraction method and continued nucleic acid amplification using PCR techniques. Primer pairs used eccB5 SK.. The positivity of DNA specific revealed amplicon in 1592 bp. PCR product was sequenced by 1st Base (First BASE Laboratories Sdn Bhd, Selangor, Malaysia). The sequence analysis used Genetyx-Win version 10.0 (Genetyx Corporation, Tokyo, Japan). RESULTS: Total isolates of Mycobacterium spp. were 28 and those that showed positive MTBC were 24 isolates and 4 nontuberculosis mycobacteria (NTM) using immunochromatographic test (ICT). The amount of homology from MTBC using blast NCBI was 99%-100%. Two SNPs were found in position c.1277 which revealed replacement of amino acid in 426 of codon position. CONCLUSION: The sequence of eccB5 gene of MTBC showed high significant homology, while proposed non-synoymous single nucleotide polymorphisms (nsSNP) may associated with clinical outcomes.