Htoo Wai - Academia.edu (original) (raw)

Papers by Htoo Wai

Research paper thumbnail of Clinical, splicing, and functional analysis to classify BRCA2 exon 3 variants: Application of a points‐based ACMG/AMP approach

Research paper thumbnail of Temporal Whole-Transcriptomic Analysis of Characterized In Vitro and Ex Vivo Primary Nasal Epithelia

Frontiers in Cell and Developmental Biology

Air-liquid interface (ALI) cell culture of primary airway progenitors enables the differentiation... more Air-liquid interface (ALI) cell culture of primary airway progenitors enables the differentiation and recapitulation of a pseudostratified epithelium in vitro, providing a highly useful tool for researching respiratory health and disease. Previous studies into gene expression in ALI-cultures compared to ex vivo nasal brushings have been limited in the number of time-points and/or the number of genes studied. In this study physiological and global transcriptomic changes were assessed in an extended in vitro 63-day human healthy nasal epithelium ALI-culture period and compared to ex vivo nasal brushing samples. Ex vivo nasal brushing samples formed distinct transcriptome clusters to in vitro ALI-cultured nasal epithelia, with from day 14 onwards ALI samples best matching the ex vivo samples. Immune response regulation genes were not expressed in the in vitro ALI-culture compared to the ex vivo nasal brushing samples, likely because the in vitro cultures lack an airway microbiome, lack...

Research paper thumbnail of Short amplicon reverse transcription‐polymerase chain reaction detects aberrant splicing in genes with low expression in blood missed by ribonucleic acid sequencing analysis for clinical diagnosis

Human Mutation

Use of blood RNA sequencing (RNA-seq) as a splicing analysis tool for clinical interpretation of ... more Use of blood RNA sequencing (RNA-seq) as a splicing analysis tool for clinical interpretation of variants of uncertain significance (VUSs) found via whole-genome and exome sequencing can be difficult for genes that have low expression in the blood due to insufficient read count coverage aligned to specific genes of interest. Here, we present a short amplicon reverse transcription-polymerase chain reaction(RT-PCR) for the detection of genes with low blood expression. Short amplicon RT-PCR, is designed to span three exons where an exon harboring a variant is flanked by one upstream and one downstream exon. We tested short amplicon RT-PCRs for genes that have median transcripts per million (TPM) values less than one according to the genotype-tissue expression database. Median TPM values of genes analyzed in this study are SYN1 = 0.8549, COL1A1 = 0.6275, TCF4 = 0.4009, DSP = .2894, TTN = 0.2851, COL5A2 = 0.1036, TERT = 0.04452, NTRK2 = 0.0344, ABCA4 = 0.00744, PRPH = 0, and WT1 = 0. All these genes show insufficient exon-spanning read coverage in our RNA-seq data to allow splicing analysis. We successfully detected all genes tested except PRPH and WT1. Aberrant splicing was detected in SYN1, TCF4, NTRK2, TTN, and TERT VUSs. Therefore, our results show short amplicon RT-PCR is a useful alternative for the analysis of splicing events in genes with low TPM in blood RNA for clinical diagnostics.

Research paper thumbnail of Additional file 1 of Biallelic variants in COPB1 cause a novel, severe intellectual disability syndrome with cataracts and variable microcephaly

Additional file 1: Fig. S1. Demonstrates how CRISPR/ cas9 genome editing induces exon skipping in... more Additional file 1: Fig. S1. Demonstrates how CRISPR/ cas9 genome editing induces exon skipping in X.tropicalis. Fig. S2. Shows the eye (a target organ) and the remainder of the crispant tadpole are equally mosaic. Fig. S3. Confirms that disruption to copb1 exon 8 in X.tropicalis mirrors syndromic hallmarks. Fig. S4. Supplemental figure demonstrates a more exaggerated phenotype in tadpoles injected with CRISPR/cas9 targeting exon 3, copb1. Fig. S5. Illustrates Xenopus anatomy and the significant reduction in brain size seen in transgenic tadpoles. Fig. S6. Shows there is no difference in localisation of wild type beta COP versus the Family 2 variant beta COP. Table S1. Details the immunodeficiency investigations performed in Family 2. Table S2. Details other shared homozygous variants identified in both probands of Family one.

Research paper thumbnail of A systematic analysis of splicing variants identifies new diagnoses in the 100,000 Genomes Project

Genomic variants which disrupt splicing are a major cause of rare genetic disease. However, varia... more Genomic variants which disrupt splicing are a major cause of rare genetic disease. However, variants which lie outside of the canonical splice sites are difficult to interpret clinically. Here, we examine the landscape of splicing variants in whole-genome sequencing data from 38,688 individuals in the 100,000 Genomes Project, and assess the contribution of non-canonical splicing variants to rare genetic diseases. We show that splicing branchpoints are highly constrained by purifying selection, and harbour damaging non-coding variants which are amenable to systematic analysis in sequencing data. From 258 de novo splicing variants in known rare disease genes, we identify 35 new likely diagnoses in probands with an unsolved rare disease. We use phenotype matching and RNA studies to confirm a new diagnosis for six individuals to date. In summary, we demonstrate the clinical value of examining non-canonical splicing variants in participants with unsolved rare diseases.

Research paper thumbnail of Transcriptome analysis of ciliary differentiation in airway epithelium

Molecular pathology and functional genomics, 2021

Research paper thumbnail of Comparison of in silico strategies to prioritize rare genomic variants impacting RNA splicing for the diagnosis of genomic disorders

Scientific Reports, 2021

The development of computational methods to assess pathogenicity of pre-messenger RNA splicing va... more The development of computational methods to assess pathogenicity of pre-messenger RNA splicing variants is critical for diagnosis of human disease. We assessed the capability of eight algorithms, and a consensus approach, to prioritize 249 variants of uncertain significance (VUSs) that underwent splicing functional analyses. The capability of algorithms to differentiate VUSs away from the immediate splice site as being ‘pathogenic’ or ‘benign’ is likely to have substantial impact on diagnostic testing. We show that SpliceAI is the best single strategy in this regard, but that combined usage of tools using a weighted approach can increase accuracy further. We incorporated prioritization strategies alongside diagnostic testing for rare disorders. We show that 15% of 2783 referred individuals carry rare variants expected to impact splicing that were not initially identified as ‘pathogenic’ or ‘likely pathogenic’; one in five of these cases could lead to new or refined diagnoses.

Research paper thumbnail of Biallelic variants in COPB1 cause a novel, severe intellectual disability syndrome with cataracts and variable microcephaly

Genome Medicine, 2021

BackgroundCoat protein complex 1 (COPI) is integral in the sorting and retrograde trafficking of ... more BackgroundCoat protein complex 1 (COPI) is integral in the sorting and retrograde trafficking of proteins and lipids from the Golgi apparatus to the endoplasmic reticulum (ER). In recent years, coat proteins have been implicated in human diseases known collectively as “coatopathies”.MethodsWhole exome or genome sequencing of two families with a neuro-developmental syndrome, variable microcephaly and cataracts revealed biallelic variants inCOPB1, which encodes the beta-subunit of COPI (β-COP). To investigate Family 1’s splice donor site variant, we undertook patient blood RNA studies and CRISPR/Cas9 modelling of this variant in a homologous region of theXenopus tropicalisgenome. To investigate Family 2’s missense variant, we studied cellular phenotypes of human retinal epithelium and embryonic kidney cell lines transfected with aCOPB1expression vector into which we had introduced Family 2’s mutation.ResultsWe present a new recessive coatopathy typified by severe developmental delay a...

Research paper thumbnail of Opposite Modulation of RAC1 by Mutations in TRIO Is Associated with Distinct, Domain-Specific Neurodevelopmental Disorders

The American Journal of Human Genetics, 2020

The Rho-guanine nucleotide exchange factor (RhoGEF) TRIO acts as a key regulator of neuronal migr... more The Rho-guanine nucleotide exchange factor (RhoGEF) TRIO acts as a key regulator of neuronal migration, axonal outgrowth, axon guidance, and synaptogenesis by activating the GTPase RAC1 and modulating actin cytoskeleton remodeling. Pathogenic variants in TRIO are associated with neurodevelopmental diseases, including intellectual disability (ID) and autism spectrum disorders (ASD). Here, we report the largest international cohort of 24 individuals with confirmed pathogenic missense or nonsense variants in TRIO. The nonsense mutations are spread along the TRIO sequence, and affected individuals show variable neurodevelopmental phenotypes. In contrast, missense variants cluster into two mutational hotspots in the TRIO sequence, one in the seventh spectrin repeat and one in the RAC1-activating GEFD1. Although all individuals in this cohort present with developmental delay and a neuro-behavioral phenotype, individuals with a pathogenic variant in the seventh spectrin repeat have a more severe ID associated with macrocephaly than do most individuals with GEFD1 variants, who display milder ID and microcephaly. Functional studies show that the spectrin and GEFD1 variants cause a TRIO-mediated hyper-or hypo-activation of RAC1, respectively, and we observe a striking correlation between RAC1 activation levels and the head size of the affected individuals. In addition, truncations in TRIO GEFD1 in the vertebrate model X. tropicalis induce defects that are concordant with the human phenotype. This work demonstrates distinct clinical and molecular disorders clustering in the GEFD1 and seventh spectrin repeat domains and highlights the importance of tight control of TRIO-RAC1 signaling in neuronal development.

Research paper thumbnail of Correction: Blood RNA analysis can increase clinical diagnostic rate and resolve variants of uncertain significance

Genetics in Medicine, 2020

An amendment to this paper has been published and can be accessed via a link at the top of the pa... more An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Research paper thumbnail of Blood RNA analysis can increase clinical diagnostic rate and resolve variants of uncertain significance

Genetics in Medicine, 2020

Purpose Diagnosis of genetic disorders is hampered by large numbers of variants of uncertain sign... more Purpose Diagnosis of genetic disorders is hampered by large numbers of variants of uncertain significance (VUSs) identified through next-generation sequencing. Many such variants may disrupt normal RNA splicing. We examined effects on splicing of a large cohort of clinically identified variants and compared performance of bioinformatic splicing prediction tools commonly used in diagnostic laboratories. Methods Two hundred fifty-seven variants (coding and noncoding) were referred for analysis across three laboratories. Blood RNA samples underwent targeted reverse transcription polymerase chain reaction (RT-PCR) analysis with Sanger sequencing of PCR products and agarose gel electrophoresis. Seventeen samples also underwent transcriptome-wide RNA sequencing with targeted splicing analysis based on Sashimi plot visualization. Bioinformatic splicing predictions were obtained using Alamut, HSF 3.1, and SpliceAI software. Results Eighty-five variants (33%) were associated with abnormal sp...

Research paper thumbnail of RNA splicing analysis in genomic medicine

The International Journal of Biochemistry & Cell Biology, 2018

High-throughput next-generation sequencing technologies have led to a rapid increase in the numbe... more High-throughput next-generation sequencing technologies have led to a rapid increase in the number of sequence variants identified in clinical practice via diagnostic genetic tests. Current bioinformatic analysis pipelines fail to take adequate account of the possible splicing effects of such variants, particularly where variants fall outwith canonical splice site sequences, and consequently the pathogenicity of such variants may often be missed. The regulation of splicing is highly complex and as a result, in silico prediction tools lack sufficient sensitivity and specificity for reliable use. Variants of all kinds can be linked to aberrant splicing in disease and the need for correct identification and diagnosis grows ever more crucial as novel splice-switching antisense oligonucleotide therapies start to enter clinical usage. RT-PCR provides a useful targeted assay of the splicing effects of identified variants, while minigene assays, massive parallel reporter assays and animal models can also be used for more detailed study of a particular splicing system, given enough time and resources. However, RNA-sequencing (RNA-seq) has the potential to be used as a rapid diagnostic tool in genomic medicine. By utilising data science approaches and machine learning, it may prove possible to finally understand and interpret the 'splicing code' and apply this knowledge in human disease diagnostics.

Research paper thumbnail of Clinical, splicing, and functional analysis to classify BRCA2 exon 3 variants: Application of a points‐based ACMG/AMP approach

Research paper thumbnail of Temporal Whole-Transcriptomic Analysis of Characterized In Vitro and Ex Vivo Primary Nasal Epithelia

Frontiers in Cell and Developmental Biology

Air-liquid interface (ALI) cell culture of primary airway progenitors enables the differentiation... more Air-liquid interface (ALI) cell culture of primary airway progenitors enables the differentiation and recapitulation of a pseudostratified epithelium in vitro, providing a highly useful tool for researching respiratory health and disease. Previous studies into gene expression in ALI-cultures compared to ex vivo nasal brushings have been limited in the number of time-points and/or the number of genes studied. In this study physiological and global transcriptomic changes were assessed in an extended in vitro 63-day human healthy nasal epithelium ALI-culture period and compared to ex vivo nasal brushing samples. Ex vivo nasal brushing samples formed distinct transcriptome clusters to in vitro ALI-cultured nasal epithelia, with from day 14 onwards ALI samples best matching the ex vivo samples. Immune response regulation genes were not expressed in the in vitro ALI-culture compared to the ex vivo nasal brushing samples, likely because the in vitro cultures lack an airway microbiome, lack...

Research paper thumbnail of Short amplicon reverse transcription‐polymerase chain reaction detects aberrant splicing in genes with low expression in blood missed by ribonucleic acid sequencing analysis for clinical diagnosis

Human Mutation

Use of blood RNA sequencing (RNA-seq) as a splicing analysis tool for clinical interpretation of ... more Use of blood RNA sequencing (RNA-seq) as a splicing analysis tool for clinical interpretation of variants of uncertain significance (VUSs) found via whole-genome and exome sequencing can be difficult for genes that have low expression in the blood due to insufficient read count coverage aligned to specific genes of interest. Here, we present a short amplicon reverse transcription-polymerase chain reaction(RT-PCR) for the detection of genes with low blood expression. Short amplicon RT-PCR, is designed to span three exons where an exon harboring a variant is flanked by one upstream and one downstream exon. We tested short amplicon RT-PCRs for genes that have median transcripts per million (TPM) values less than one according to the genotype-tissue expression database. Median TPM values of genes analyzed in this study are SYN1 = 0.8549, COL1A1 = 0.6275, TCF4 = 0.4009, DSP = .2894, TTN = 0.2851, COL5A2 = 0.1036, TERT = 0.04452, NTRK2 = 0.0344, ABCA4 = 0.00744, PRPH = 0, and WT1 = 0. All these genes show insufficient exon-spanning read coverage in our RNA-seq data to allow splicing analysis. We successfully detected all genes tested except PRPH and WT1. Aberrant splicing was detected in SYN1, TCF4, NTRK2, TTN, and TERT VUSs. Therefore, our results show short amplicon RT-PCR is a useful alternative for the analysis of splicing events in genes with low TPM in blood RNA for clinical diagnostics.

Research paper thumbnail of Additional file 1 of Biallelic variants in COPB1 cause a novel, severe intellectual disability syndrome with cataracts and variable microcephaly

Additional file 1: Fig. S1. Demonstrates how CRISPR/ cas9 genome editing induces exon skipping in... more Additional file 1: Fig. S1. Demonstrates how CRISPR/ cas9 genome editing induces exon skipping in X.tropicalis. Fig. S2. Shows the eye (a target organ) and the remainder of the crispant tadpole are equally mosaic. Fig. S3. Confirms that disruption to copb1 exon 8 in X.tropicalis mirrors syndromic hallmarks. Fig. S4. Supplemental figure demonstrates a more exaggerated phenotype in tadpoles injected with CRISPR/cas9 targeting exon 3, copb1. Fig. S5. Illustrates Xenopus anatomy and the significant reduction in brain size seen in transgenic tadpoles. Fig. S6. Shows there is no difference in localisation of wild type beta COP versus the Family 2 variant beta COP. Table S1. Details the immunodeficiency investigations performed in Family 2. Table S2. Details other shared homozygous variants identified in both probands of Family one.

Research paper thumbnail of A systematic analysis of splicing variants identifies new diagnoses in the 100,000 Genomes Project

Genomic variants which disrupt splicing are a major cause of rare genetic disease. However, varia... more Genomic variants which disrupt splicing are a major cause of rare genetic disease. However, variants which lie outside of the canonical splice sites are difficult to interpret clinically. Here, we examine the landscape of splicing variants in whole-genome sequencing data from 38,688 individuals in the 100,000 Genomes Project, and assess the contribution of non-canonical splicing variants to rare genetic diseases. We show that splicing branchpoints are highly constrained by purifying selection, and harbour damaging non-coding variants which are amenable to systematic analysis in sequencing data. From 258 de novo splicing variants in known rare disease genes, we identify 35 new likely diagnoses in probands with an unsolved rare disease. We use phenotype matching and RNA studies to confirm a new diagnosis for six individuals to date. In summary, we demonstrate the clinical value of examining non-canonical splicing variants in participants with unsolved rare diseases.

Research paper thumbnail of Transcriptome analysis of ciliary differentiation in airway epithelium

Molecular pathology and functional genomics, 2021

Research paper thumbnail of Comparison of in silico strategies to prioritize rare genomic variants impacting RNA splicing for the diagnosis of genomic disorders

Scientific Reports, 2021

The development of computational methods to assess pathogenicity of pre-messenger RNA splicing va... more The development of computational methods to assess pathogenicity of pre-messenger RNA splicing variants is critical for diagnosis of human disease. We assessed the capability of eight algorithms, and a consensus approach, to prioritize 249 variants of uncertain significance (VUSs) that underwent splicing functional analyses. The capability of algorithms to differentiate VUSs away from the immediate splice site as being ‘pathogenic’ or ‘benign’ is likely to have substantial impact on diagnostic testing. We show that SpliceAI is the best single strategy in this regard, but that combined usage of tools using a weighted approach can increase accuracy further. We incorporated prioritization strategies alongside diagnostic testing for rare disorders. We show that 15% of 2783 referred individuals carry rare variants expected to impact splicing that were not initially identified as ‘pathogenic’ or ‘likely pathogenic’; one in five of these cases could lead to new or refined diagnoses.

Research paper thumbnail of Biallelic variants in COPB1 cause a novel, severe intellectual disability syndrome with cataracts and variable microcephaly

Genome Medicine, 2021

BackgroundCoat protein complex 1 (COPI) is integral in the sorting and retrograde trafficking of ... more BackgroundCoat protein complex 1 (COPI) is integral in the sorting and retrograde trafficking of proteins and lipids from the Golgi apparatus to the endoplasmic reticulum (ER). In recent years, coat proteins have been implicated in human diseases known collectively as “coatopathies”.MethodsWhole exome or genome sequencing of two families with a neuro-developmental syndrome, variable microcephaly and cataracts revealed biallelic variants inCOPB1, which encodes the beta-subunit of COPI (β-COP). To investigate Family 1’s splice donor site variant, we undertook patient blood RNA studies and CRISPR/Cas9 modelling of this variant in a homologous region of theXenopus tropicalisgenome. To investigate Family 2’s missense variant, we studied cellular phenotypes of human retinal epithelium and embryonic kidney cell lines transfected with aCOPB1expression vector into which we had introduced Family 2’s mutation.ResultsWe present a new recessive coatopathy typified by severe developmental delay a...

Research paper thumbnail of Opposite Modulation of RAC1 by Mutations in TRIO Is Associated with Distinct, Domain-Specific Neurodevelopmental Disorders

The American Journal of Human Genetics, 2020

The Rho-guanine nucleotide exchange factor (RhoGEF) TRIO acts as a key regulator of neuronal migr... more The Rho-guanine nucleotide exchange factor (RhoGEF) TRIO acts as a key regulator of neuronal migration, axonal outgrowth, axon guidance, and synaptogenesis by activating the GTPase RAC1 and modulating actin cytoskeleton remodeling. Pathogenic variants in TRIO are associated with neurodevelopmental diseases, including intellectual disability (ID) and autism spectrum disorders (ASD). Here, we report the largest international cohort of 24 individuals with confirmed pathogenic missense or nonsense variants in TRIO. The nonsense mutations are spread along the TRIO sequence, and affected individuals show variable neurodevelopmental phenotypes. In contrast, missense variants cluster into two mutational hotspots in the TRIO sequence, one in the seventh spectrin repeat and one in the RAC1-activating GEFD1. Although all individuals in this cohort present with developmental delay and a neuro-behavioral phenotype, individuals with a pathogenic variant in the seventh spectrin repeat have a more severe ID associated with macrocephaly than do most individuals with GEFD1 variants, who display milder ID and microcephaly. Functional studies show that the spectrin and GEFD1 variants cause a TRIO-mediated hyper-or hypo-activation of RAC1, respectively, and we observe a striking correlation between RAC1 activation levels and the head size of the affected individuals. In addition, truncations in TRIO GEFD1 in the vertebrate model X. tropicalis induce defects that are concordant with the human phenotype. This work demonstrates distinct clinical and molecular disorders clustering in the GEFD1 and seventh spectrin repeat domains and highlights the importance of tight control of TRIO-RAC1 signaling in neuronal development.

Research paper thumbnail of Correction: Blood RNA analysis can increase clinical diagnostic rate and resolve variants of uncertain significance

Genetics in Medicine, 2020

An amendment to this paper has been published and can be accessed via a link at the top of the pa... more An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Research paper thumbnail of Blood RNA analysis can increase clinical diagnostic rate and resolve variants of uncertain significance

Genetics in Medicine, 2020

Purpose Diagnosis of genetic disorders is hampered by large numbers of variants of uncertain sign... more Purpose Diagnosis of genetic disorders is hampered by large numbers of variants of uncertain significance (VUSs) identified through next-generation sequencing. Many such variants may disrupt normal RNA splicing. We examined effects on splicing of a large cohort of clinically identified variants and compared performance of bioinformatic splicing prediction tools commonly used in diagnostic laboratories. Methods Two hundred fifty-seven variants (coding and noncoding) were referred for analysis across three laboratories. Blood RNA samples underwent targeted reverse transcription polymerase chain reaction (RT-PCR) analysis with Sanger sequencing of PCR products and agarose gel electrophoresis. Seventeen samples also underwent transcriptome-wide RNA sequencing with targeted splicing analysis based on Sashimi plot visualization. Bioinformatic splicing predictions were obtained using Alamut, HSF 3.1, and SpliceAI software. Results Eighty-five variants (33%) were associated with abnormal sp...

Research paper thumbnail of RNA splicing analysis in genomic medicine

The International Journal of Biochemistry & Cell Biology, 2018

High-throughput next-generation sequencing technologies have led to a rapid increase in the numbe... more High-throughput next-generation sequencing technologies have led to a rapid increase in the number of sequence variants identified in clinical practice via diagnostic genetic tests. Current bioinformatic analysis pipelines fail to take adequate account of the possible splicing effects of such variants, particularly where variants fall outwith canonical splice site sequences, and consequently the pathogenicity of such variants may often be missed. The regulation of splicing is highly complex and as a result, in silico prediction tools lack sufficient sensitivity and specificity for reliable use. Variants of all kinds can be linked to aberrant splicing in disease and the need for correct identification and diagnosis grows ever more crucial as novel splice-switching antisense oligonucleotide therapies start to enter clinical usage. RT-PCR provides a useful targeted assay of the splicing effects of identified variants, while minigene assays, massive parallel reporter assays and animal models can also be used for more detailed study of a particular splicing system, given enough time and resources. However, RNA-sequencing (RNA-seq) has the potential to be used as a rapid diagnostic tool in genomic medicine. By utilising data science approaches and machine learning, it may prove possible to finally understand and interpret the 'splicing code' and apply this knowledge in human disease diagnostics.