From bedside to genetic analysis: New insights into pathophysiology of melanoma, basal cell carcinoma, and other cancers.

OBJECTIVE: Patients with myotonic muscular dystrophy (MMD) were observed to have numerous basal cell carcinoma (BCC) and abnormal dysplastic nevi (DN) on non-sun exposed skin. Simultaneously a large study published in the Journal of American Medical Association (JAMA) illustrated that patients with MMD have "overall" an increased risk for cancer development. Based on these findings, this author in 2010 postulated that dysregulation of RNA binding proteins (RBP), responsible for clinical manifestations of MMD, is also responsible for the development of BCC and melanoma. METHODS: To report new research elucidating the etiology of melanoma, BCC, MMD-induced cancers, and potentially other environmentally induced malignancies. RESULTS: Dysregulation of RBP induces aberrant mRNA splicing; recent data indicates that abnormal mRNA splicing not just plays a key role in the pathogenesis of melanoma but is a hallmark of essentially all human malignancies. CONCLUSION: The author's hypothesis is that ultraviolet (UV) radiation induces DNA damage in intronic regions of a variety of genes. Furthermore, these UV-induced abnormal DNA dimers, repeats and mutations interfere with normal mRNA splicing thus producing abnormal proteins. These abnormal proteins in turn activate oncogenic pathways such as hedgehog, MAP kinase, and WNT.

Cryptic exon activation caused by a novel deep-intronic splice-altering variant in Becker muscular dystrophy.

BACKGROUND: An accurate genetic diagnosis of Becker muscular dystrophy (BMD) can be sometimes challenging due to deep intronic DMD variants. Here, we report on the genetic diagnosis of a BMD patient with a novel deep-intronic splice-altering variant in DMD. METHODS: The index case was a 3.8-year-old boy who was suspected of having a diagnosis of BMD based on his clinical, muscle imaging, and pathological features. Routine genomic detection approaches did not detect any disease-causing variants in him. Muscle-derived DMD mRNA studies, followed by genomic Sanger sequencing and in silico bioinformatic analyses, were performed in the patient. RESULTS: DMD mRNA studies detected a cryptic exon-containing transcript and normally spliced DMD transcript in the patient. The cryptic exon-containing transcript encoded a frameshift and premature termination codon (NP_003997.1:p.[=,Asp2740Valfs*52]). Further genomic Sanger sequencing and bioinformatic analysis identified a novel deep-intronic splice-altering variant in DMD (c.8217 + 23338A > G). The novel variant strengthened a cryptic donor splice site and activated a cryptic acceptor splice site in the deep-intronic region of DMD intron 55, resulting in the activation of a new dystrophin cryptic exon found in the patient. CONCLUSION: Our case report expands the genetic spectrum of BMD and highlights the essential role of deep-intronic cryptic exon-activating variants in genetically unsolved BMD patients.

Bi-allelic loss-of-function variants in WBP4, encoding a spliceosome protein, result in a variable neurodevelopmental syndrome.

Over two dozen spliceosome proteins are involved in human diseases, also referred to as spliceosomopathies. WW domain-binding protein 4 (WBP4) is part of the early spliceosomal complex and has not been previously associated with human pathologies in the Online Mendelian Inheritance in Man (OMIM) database. Through GeneMatcher, we identified ten individuals from eight families with a severe neurodevelopmental syndrome featuring variable manifestations. Clinical manifestations included hypotonia, global developmental delay, severe intellectual disability, brain abnormalities, musculoskeletal, and gastrointestinal abnormalities. Genetic analysis revealed five different homozygous loss-of-function variants in WBP4. Immunoblotting on fibroblasts from two affected individuals with different genetic variants demonstrated a complete loss of protein, and RNA sequencing analysis uncovered shared abnormal splicing patterns, including in genes associated with abnormalities of the nervous system, potentially underlying the phenotypes of the probands. We conclude that bi-allelic variants in WBP4 cause a developmental disorder with variable presentations, adding to the growing list of human spliceosomopathies.

Code inside the codon: The role of synonymous mutations in regulating splicing machinery and its impact on disease.

In eukaryotes, precise pre-mRNA processing, including alternative splicing, is essential to carry out the intricate protein translation process. Both point mutations (that alter the translated protein sequence) and synonymous mutations (that do not alter the translated protein sequence) are capable of affecting the splicing process. Synonymous mutations are known to affect gene expression via altering mRNA stability, mRNA secondary structure, splicing processes, and translational kinetics. In higher eukaryotes, precise splicing is regulated by three weakly conserved cis-elements, 5' and 3' splice sites and the branch site. Many other cis-acting elements (exonic/intronic splicing enhancers and silencers) and trans-acting splicing factors (serine and arginine-rich proteins and heterogeneous nuclear ribonucleoproteins) have also been found to enhance or suppress the splicing process. The appearance of synonymous mutations in cis-acting elements can alter the splicing process by changing the binding pattern of splicing factors to exonic splicing enhancers or silencer motifs. This results in exon skipping, intron retention, and various other forms of alternative splicing, eventually leading to the emergence of a wide range of diseases. The focus of this review is to elucidate the role of synonymous mutations and their impact on abnormal splicing mechanisms. Further, this study highlights the function of synonymous mutation in mediating abnormal splicing in cancer and development of X-linked, and autosomal inherited diseases.

Computational prediction of human deep intronic variation.

BACKGROUND: The adoption of whole-genome sequencing in genetic screens has facilitated the detection of genetic variation in the intronic regions of genes, far from annotated splice sites. However, selecting an appropriate computational tool to discriminate functionally relevant genetic variants from those with no effect is challenging, particularly for deep intronic regions where independent benchmarks are scarce. RESULTS: In this study, we have provided an overview of the computational methods available and the extent to which they can be used to analyze deep intronic variation. We leveraged diverse datasets to extensively evaluate tool performance across different intronic regions, distinguishing between variants that are expected to disrupt splicing through different molecular mechanisms. Notably, we compared the performance of SpliceAI, a widely used sequence-based deep learning model, with that of more recent methods that extend its original implementation. We observed considerable differences in tool performance depending on the region considered, with variants generating cryptic splice sites being better predicted than those that potentially affect splicing regulatory elements. Finally, we devised a novel quantitative assessment of tool interpretability and found that tools providing mechanistic explanations of their predictions are often correct with respect to the ground - information, but the use of these tools results in decreased predictive power when compared to black box methods. CONCLUSIONS: Our findings translate into practical recommendations for tool usage and provide a reference framework for applying prediction tools in deep intronic regions, enabling more informed decision-making by practitioners.

Transcriptome Analysis Reveals Higher Levels of Mobile Element-Associated Abnormal Gene Transcripts in Temporal Lobe Epilepsy Patients.

Mesial temporal lobe epilepsy (MTLE) is the most common form of epilepsy, and temporal lobe epilepsy patients with hippocampal sclerosis (TLE-HS) show worse drug treatment effects and prognosis. TLE has been shown to have a genetic component, but its genetic research has been mostly limited to coding sequences of genes with known association to epilepsy. Representing a major component of the genome, mobile elements (MEs) are believed to contribute to the genetic etiology of epilepsy despite limited research. We analyzed publicly available human RNA-seq-based transcriptome data to determine the role of mobile elements in epilepsy by performing de novo transcriptome assembly, followed by identification of spliced gene transcripts containing mobile element (ME) sequences (ME-transcripts), to compare their frequency across different sample groups. Significantly higher levels of ME-transcripts in hippocampal tissues of epileptic patients, particularly in TLE-HS, were observed. Among ME classes, short interspersed nuclear elements (SINEs) were shown to be the most frequent contributor to ME-transcripts, followed by long interspersed nuclear elements (LINEs) and DNA transposons. These ME sequences almost in all cases represent older MEs normally located in the intron sequences. For protein coding genes, ME sequences were mostly found in the 3'-UTR regions, with a significant portion also in the coding sequences (CDSs), leading to reading frame disruption. Genes associated with ME-transcripts showed enrichment for the mRNA splicing process and an apparent bias in epileptic transcriptomes toward neural- and epilepsy-associated genes. The findings of this study suggest that abnormal splicing involving MEs, leading to loss of functions in critical genes, plays a role in epilepsy, particularly in TLE-HS, thus providing a novel insight into the molecular mechanisms underlying epileptogenesis.

Pathogenic Abnormal Splicing Due to Intronic Deletions that Induce Biophysical Space Constraint for Spliceosome Assembly.

A precise genetic diagnosis is the single most important step for families with genetic disorders to enable personalized and preventative medicine. In addition to genetic variants in coding regions (exons) that can change a protein sequence, abnormal pre-mRNA splicing can be devastating for the encoded protein, inducing a frameshift or in-frame deletion/insertion of multiple residues. Non-coding variants that disrupt splicing are extremely challenging to identify. Stemming from an initial clinical discovery in two index Australian families, we define 25 families with genetic disorders caused by a class of pathogenic non-coding splice variant due to intronic deletions. These pathogenic intronic deletions spare all consensus splice motifs, though they critically shorten the minimal distance between the 5' splice-site (5'SS) and branchpoint. The mechanistic basis for abnormal splicing is due to biophysical constraint precluding U1/U2 spliceosome assembly, which stalls in A-complexes (that bridge the 5'SS and branchpoint). Substitution of deleted nucleotides with non-specific sequences restores spliceosome assembly and normal splicing, arguing against loss of an intronic element as the primary causal basis. Incremental lengthening of 5'SS-branchpoint length in our index EMD case subject defines 45-47 nt as the critical elongation enabling (inefficient) spliceosome assembly for EMD intron 5. The 5'SS-branchpoint space constraint mechanism, not currently factored by genomic informatics pipelines, is relevant to diagnosis and precision medicine across the breadth of Mendelian disorders and cancer genomics.

Abnormalities in Alternative Splicing of Apoptotic Genes and Cardiovascular Diseases.

Apoptosis is required for normal heart development in the embryo, but has also been shown to be an important factor in the occurrence of heart disease. Alternative splicing of apoptotic genes is currently emerging as a diagnostic and therapeutic target for heart disease. This review addresses the involvement of abnormalities in alternative splicing of apoptotic genes in cardiac disorders including cardiomyopathy, myocardial ischemia and heart failure. Many pro-apoptotic members of the Bcl-2 family have alternatively spliced isoforms that lack important active domains. These isoforms can play a negative regulatory role by binding to and inhibiting the pro-apoptotic forms. Alternative splicing is observed to be increased in various cardiovascular diseases with the level of alternate transcripts increasing elevated in diseased hearts compared to healthy subjects. In many cases these isoforms appear to be the underlying cause of the disease, while in others they may be induced in response to cardiovascular pathologies. Regardless of this, the detection of alternate splicing events in the heart can serve as useful diagnostic or prognostic tools, while those splicing events that seem to play a causative role in cardiovascular disease make attractive future drug targets.

Abnormal splicing in the N-terminal variable region of cardiac troponin T impairs systolic function of the heart with preserved Frank-Starling compensation.

Abnormal splice-out of the exon 7-encoded segment in the N-terminal variable region of cardiac troponin T (cTnT-ΔE7) was found in turkeys and, together with the inclusion of embryonic exon (eTnT), in adult dogs with a correlation with dilated cardiomyopathy. Overexpression of these cTnT variants in transgenic mouse hearts significantly decreased cardiac function. To further investigate the functional effect of cTnT-ΔE7 or ΔE7+eTnT in vivo under systemic regulation, echocardiography was carried out in single and double-transgenic mice. No atrial enlargement, ventricular hypertrophy or dilation was detected in the hearts of 2-month-old cTnT-ΔE7 and ΔE7+eTnT mice in comparison to wild-type controls, indicating a compensated state. However, left ventricular fractional shortening and ejection fraction were decreased in ΔE7 and ΔE7+eTnT mice, and the response to isoproterenol was lower in ΔE7+eTnT mice. Left ventricular outflow tract velocity and gradient were decreased in the transgenic mouse hearts, indicating decreased systolic function. Ex vivo working heart function showed that high afterload or low preload resulted in more severe decreases in the systolic function and energetic efficiency of cTnT-ΔE7 and ΔE7+eTnT hearts. On the other hand, increases in preload demonstrated preserved Frank-Starling responses and minimized the loss of cardiac function and efficiency. The data demonstrate that the N-terminal variable region of cardiac TnT regulates systolic function of the heart.

UBE2B mRNA alterations are associated with severe oligozoospermia in infertile men.

Oligozoospermia (low sperm count) is a common semen deficiency. However, to date, few genetic defects have been identified to cause this condition. Moreover, even fewer molecular genetic diagnostic tests are available for patients with oligozoospermia in the andrology clinic. Based on animal and gene expression studies of oligozoospermia, several molecular pathways may be disrupted in post-meiotic spermatozoa. One of the disrupted pathways is protein ubiquitination and cell apoptosis. A critical protein involved in this pathway is the ubiquitin-conjugating enzyme 2B, UBE2B. Absence of Ube2b in male mice causes spermatogenic meiotic disruption with increased apoptosis, leading to infertility. To examine the association between messenger RNA defects in UBE2B and severe oligozoospermia (0.1-10 × 10(6) cells/ml), sequencing of sperm cDNA in 326 oligozoospermic patients and 421 normozoospermic men was performed. mRNA alterations in UBE2B were identified in sperm in 4.6% (15 out of 326) of the oligozoospermic patients, but not found in control men, suggesting strong association between mRNA defects and oligozoospermia (χ(2) = 19, P = 0.0001). Identified UBE2B alterations include nine splicing, four missense and two nonsense alterations. The follow-up screen of corresponding DNA regions did not reveal causative DNA mutations, suggesting a post-transcriptional nature of identified defects. None of these variants were reported in the dbSNP database, although other splicing abnormalities with low level of expression were present in 11 out of 421 (2.6%) controls. Our findings suggest that two distinct molecular mechanisms, mRNA editing and splicing processing, are disrupted in oligozoospermia. We speculate that the contribution of post-transcriptional mRNA defects to oligozoospermia could be greater than previously anticipated.