Identification of Genomic Alterations in Thai Patients With Colorectal Cancer Using Next-Generation Sequencing-Based Multigene Cancer Panel.

Introduction Colorectal cancer (CRC) is one of the leading causes of death and illness in the general population. Although the incidence of CRC is steadily decreasing worldwide, it is being diagnosed more in individuals under 50 years of age. Multiple disease-causing variants have been reported to be involved in the development of CRC. This study aimed to investigate the molecular and clinical characteristics of Thai patients with CRC. Methods NGS-based multigene cancer panel testing was performed on 21 unrelated patients. Target enrichment was performed using a custom-designed Ion AmpliSeq on-demand panel. Thirty-six genes associated with CRC and other cancer were analyzed for variant detection. Results Sixteen variants (five nonsense, eight missense, two deletions, and one duplication) in nine genes were identified in 12 patients. Eight (66.7%) patients harboring disease-causing deleterious variants in genes APC, ATM, BRCA2, MSH2, and MUTYH. One of the eight patients also carried additional heterozygous variants in genes ATM, BMPR1A, and MUTYH. In addition, four patients carried variants of uncertain significance in genes APC, MLH1, MSH2, STK11, and TP53. Among all detected genes, APC was the most frequent causative gene observed in CRC patients, which is consistent with previous reports. Conclusion This study demonstrated the comprehensive molecular and clinical characterization of CRC patients. These findings showed the benefits of using multigene cancer panel sequencing for pathogenic gene detection and showed the prevalence of genetic aberrations in Thai patients with CRC.

Genotypic and phenotypic profiles of EYS gene-related retinitis pigmentosa: a retrospective study.

Retinitis pigmentosa (RP) affects 1:5000 individuals worldwide. Interestingly, variations in 271 RP-related genes are indicated to vary among populations. We aimed to evaluate the genetic prevalence and phenotypic profiles of Thai patients with RP. The clinical and whole exome sequencing data of 125 patients suggestive of inherited retinal diseases (IRD), particularly non-syndromic RP, were assessed. We found a total of 258 variants (63% of which remained unavailable in the ClinVar database) in 91 IRD-associated genes. Among the detected genes, the eyes shut homolog (EYS) gene showed the highest prevalence. We also provide insights into the genotypic, baseline, and follow-up clinical presentations of seven patients with disease-causing EYS variations. This study could provide comprehension of the prevalence of RP-related genes involved in the Asian population. It might also provide information to establish advanced and personalised therapy for RP in the Thai population.

Molecular and clinical characterization of Thai patients with achromatopsia: identification of three novel disease-associated variants in the CNGA3 and CNGB3 genes.

PURPOSE: Achromatopsia (ACHM) is an autosomal recessive cone disorder characterized by pendular nystagmus, photophobia, reduced visual acuity, and partial or total absence of color vision. Mutations in six genes (CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, and ATF6) have been reported in ACHM. There is no information on these disease-associated genes in Thai population. This study aimed to investigate the molecular and clinical characteristics in Thai patients with ACHM. METHODS: Seven unrelated Thai patients with ACHM were recruited. Detailed ophthalmologic examination was performed. Polymerase chain reaction (PCR)-coupled single-strand conformation polymorphism (SSCP) screening followed by Sanger sequencing was used to identify sequence variants in all exons and splice junctions of three genes (CNGA3, CNGB3, and GNAT2). The pathogenicity of the detected variants was interpreted. Segregation analysis was performed to determine variant sharing in available family members. RESULTS: Four patients displayed different SSCP migration patterns. Sequence analysis revealed a reported pathogenic and a novel disease-associated variant in the CNGA3 gene. For the CNGB3 gene, we found two novel disease-associated variants and a reported variant of uncertain significance (VUS). Segregation analysis confirmed that the variants identified in each patient were present in the heterozygous state in their corresponding family members, which was consistent with an autosomal recessive mode of inheritance. CONCLUSIONS: This study demonstrated the first molecular and clinical characterization of ACHM in Thai patients. The identification of disease-associated genes in a specific population leads to a personalized gene therapy benefiting those affected patients.

Tumor mutational profile of triple negative breast cancer patients in Thailand revealed distinctive genetic alteration in chromatin remodeling gene.

BACKGROUND: Triple negative breast cancer (TNBC) is a breast cancer subtype characterized by absence of both hormonal receptors and human epithelial growth factor receptor 2 (HER2). TNBC accounts for 15-20% of breast cancer. TNBC is associated with more aggressive disease and worse clinical outcome. Though the underlying mechanism of TNBC is currently unclear, the heterogeneity of clinical characteristics in various population may relate to the difference in tumor mutational profile. There were studies on TNBC gene mutations in various ethnic groups but the tumor genome data on Thai TNBC patients is currently unknown. This study aims to investigate mutational profile of Thai TNBC. METHODS: The patients were Thai individuals who were diagnosed with primary breast carcinoma between 2014 and 2017. All surgically removed primary tumor tissues were carefully examined by pathologists and archived as formalin-fixed paraffin-embedded tumor. TNBC was defined by absence of hormonal receptors and HER2 by immunohistochemistry. Genomic DNA was extracted, enriched and sequenced of all exomes on the Illumina HiSeq. Genomic data were then processed through bioinformatics platform to identify genomic alterations and tumor mutational burden. RESULTS: A total of 116 TNBC patients were recruited. Genomic analysis of TNBC samples identified 81,460 variants, of which 5,906 variants were in cancer-associated genes. The result showed that Thai TNBC has higher tumor mutation burden than previously reported data. The most frequently mutated cancer-associated gene was TP53 similar to other TNBC cohorts. Meanwhile KMT2C was found to be more commonly mutated in Thai TNBC than previous studies. Mutational profile of Thai TNBC patients also revealed difference in many frequently mutated genes when compared to other Western TNBC cohorts. CONCLUSION: This result supported that TNBC breast cancer patients from various ethnic background showed diverse genome alteration pattern. Although TP53 is the most commonly mutated gene across all cohorts, Thai TNBC showed different gene mutation frequencies, especially in KMT2C. In particular, the cancer gene mutations are more prevalent in Thai TNBC patients. This result provides important insight on diverse underlying genetic and epigenetic mechanisms of TNBC that could translate to a new treatment strategy for patients with this disease.

Whole Exome Sequencing in Eight Thai Patients With Leber Congenital Amaurosis Reveals Mutations in the CTNNA1 and CYP4V2 Genes.

Purpose: Our goal was to describe the clinical and molecular genetic findings in Thai patients with Leber congenital amaurosis (LCA). Methods: Whole exome sequencing (WES) was performed in eight unrelated patients. All genes responsible for inherited retinal diseases (IRDs) based on RetNet were selected for analysis. Potentially causative variants were filtered through a bioinformatics pipeline and validated using Sanger sequencing. Segregation analysis of the causative genes was performed in family members when available. Results: Eleven deleterious variants, six nonsense and five missense, were identified in seven genes: four LCA-associated genes (CEP290, IQCB1, NMNAT1, and RPGRIP1), one gene responsible for syndromic LCA (ALMS1), and two IRDs-related genes (CTNNA1 and CYP4V2). Clinical reassessment supported the diagnosis of syndromic LCA in those patients harboring potentially pathogenic variants in the ALMS1. Interestingly, two causative genes, CTNNA1 and CYP4V2, previously reported to cause butterfly-shaped pigment dystrophy (BSPD) and Bietti's crystalline dystrophy (BCD), respectively, were detected in two other patients. These two patients developed rapid and severe visual loss in contrast to BSPD and BCD patients in previous studies. The results of this study demonstrate that causative variants identified in the CTNNA1 and CYP4V2 genes are also associated with LCA. Conclusions: This is the first report describing the molecular genetics and clinical manifestations of Thai patients with LCA. The present study expands the spectrum of LCA-associated genes, which is a benefit for molecular diagnosis. The identification of mutations in the CTNNA1 and CYP4V2 genes requires further elucidation in larger cohorts with LCA.

A novel start codon mutation of the MERTK gene in a patient with retinitis pigmentosa.

PURPOSE: Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of inherited retinal degenerations characterized by progressive loss of photoreceptor cells and RPE functions. More than 70 causative genes are known to be responsible for RP. This study aimed to identify the causative gene in a patient from a consanguineous family with childhood-onset severe retinal dystrophy. METHODS: To identify the defective gene, whole exome sequencing was performed. Candidate causative variants were selected and validated using Sanger sequencing. Segregation analysis of the causative gene was performed in additional family members. To verify that the mutation has an effect on protein synthesis, an expression vector containing the first ten amino acids of the mutant protein fused with the DsRed2 fluorescent protein was constructed and transfected into HEK293T cells. Expression of the fusion protein in the transfected cells was measured using fluorescence microscopy. RESULTS: By filtering against public variant databases, a novel homozygous missense mutation (c.3G>A) localized in the start codon of the MERTK gene was detected as a potentially pathogenic mutation for autosomal recessive RP. The c.3G>A mutation cosegregated with the disease phenotype in the family. No expression of the first ten amino acids of the MerTK mutant fused with the DsRed2 fluorescent protein was detected in HEK293T cells, indicating that the mutation affects the translation initiation site of the gene that may lead to loss of function of the MerTK signaling pathway. CONCLUSIONS: We report a novel missense mutation (c.3G>A, p.0?) in the MERTK gene that causes severe vision impairment in a patient. Taken together with previous reports, our results expand the spectrum of MERTK mutations and extend our understanding of the role of the MerTK protein in the pathogenesis of retinitis pigmentosa.

Whole exome sequencing in Thai patients with retinitis pigmentosa reveals novel mutations in six genes.

PURPOSE: To identify disease-causing mutations and describe genotype-phenotype correlations in Thai patients with nonsyndromic retinitis pigmentosa (RP). METHODS: Whole exome sequencing was performed in 20 unrelated patients. Eighty-six genes associated with RP, Leber congenital amaurosis, and cone-rod dystrophy were analyzed for variant detection. RESULTS: Seventeen variants (13 novel and 4 known) in 13 genes were identified in 11 patients. These variants include 10 missense substitutions, 2 nonsense mutations, 3 deletions, 1 insertion, and 1 splice site change. Nine patients with identified inheritance patterns carried a total of 10 potentially pathogenic mutations located in genes CRB1, C8orf37, EYS, PROM1, RP2, and USH2A. Three of the nine patients also demonstrated additional heterozygous variants in genes ABCA4, GUCY2D, RD3, ROM1, and TULP1. In addition, two patients carried variants of uncertain significance in genes FSCN2 and NR2E3. The RP phenotypes of our patients were consistent with previous reports. CONCLUSIONS: This is the first report of mutations in Thai RP patients. These findings are useful for genotype-phenotype comparisons among different ethnic groups.

Run-on mutation in the PAX6 gene and chorioretinal degeneration in autosomal dominant aniridia.

PURPOSE: To identify the causative paired box 6 (PAX6) mutation in a family with autosomal dominant aniridia. METHODS: A family with autosomal dominant aniridia with three affected individuals in two generations was investigated for the causative PAX6 mutation by single strand conformation polymorphism (SSCP) followed by sequencing of genomic DNA from peripheral blood. RESULTS: A novel PAX6 mutation in the donor splice site of intron 12 was identified in all three affected individuals from the family. The automated splice site analysis web interface indicated a disturbance of splicing and it was predicted that this mutation could lead to an elimination of the normal stop codon and an abnormal 3' elongation of the mRNA. CONCLUSIONS: We report a novel PAX6 mutation in autosomal dominant aniridia that presumably affects splicing. The presence of chorioretinal degeneration in one of the affected individual raises the possibility that run-on mutations are associated with chorioretinal involvement in aniridia.

Mutations in the XLRS1 gene in Thai families with X-linked juvenile retinoschisis.

PURPOSE: To identify genetic mutations of the XLRS1 gene and to describe the ocular phenotypes in two unrelated Thai patients with X-linked juvenile retinoschisis. METHODS: Ophthalmic examination, including best-corrected visual acuity and fundus examination and photography, was performed in all participants. Electroretinography (ERG) and optical coherence tomography were performed when possible. All six exons of the XLRS1 gene were amplified, and mutation screening was determined by denaturing high-performance liquid chromatography and DNA sequencing. RESULTS: Two point mutations were identified, a novel missense mutation c.378A > G (p.D126G) in exon 5 and a reported mutation c.637C > T (p.R213W) in exon 6. The first proband with the p.D126G mutation developed vitreous hemorrhage in both eyes at age 7 months. Foveal and peripheral schisis with several inner layer holes were detected in both eyes. The second proband with the p.R213W mutation developed slightly blurred vision at age 10 years. Fundus examination showed numerous fine white dots at the macula without foveal or peripheral schisis. Electronegative ERG results were documented in both probands. CONCLUSIONS: A novel p.D126G mutation appeared to be associated with a severe phenotype with vitreous hemorrhage developing in infancy. Both intra- and interfamilial clinical variabilities were recognized in our patients.

Mutation analysis of the VMD2 gene in thai families with best macular dystrophy.

BACKGROUND: To identify genetic mutations of the VMD2 gene in two Thai families with Best macular dystrophy. MATERIALS AND METHODS: Ophthalmic examination including best-corrected visual acuity (BCVA), dilated fundus examination and fundus photography, and electro-oculography (EOG) was performed in two probands and their family members. Mutation screening of the VMD2 gene was performed by single-strand conformation polymorphism (SSCP) analysis followed by direct DNA sequencing of the abnormal exons. RESULTS: The 58-year-old male proband demonstrated typical egg yolk-like macular lesion in both eyes. Mutational screening of VMD2 identified a band shift in exon 7, which was confirmed by direct DNA sequencing to be a G to A transition at position 724 bp. This novel missense mutation resulted in the change of an amino acid valine to methionine and was responsible for the abnormal Arden ratio in the proband's daughter. The second male proband age 25 had a characteristic egg yolk-like macular lesion in the left eye and a scrambled egg appearance in the right. Mutation analysis identified a C to T transition at position 652 bp in exon 6. This reported missense mutation led to an amino acid substitution of cysteine for arginine. The mutation was documented in the maternal grandmother, the mother, as well as the elder sister of the proband. CONCLUSIONS: The Val-242-Met mutation is associated with a late-onset visual disturbance and the Arg-218-Cys mutation was associated with marked intra-familial clinical variability of expression. Presymptomatic testing will be available to the family members at risk with high accuracy.