Somatic symptom disorder

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https://en.wikipedia.org/wiki/Somatic_symptom_disorder 

 

Somatic symptom disorder
Other names	Somatoform disorder
Specialty	Psychiatry, Psychology
Symptoms	Maladaptive thoughts, feelings, and behaviors in response to chronic physical symptoms.
Complications	Reduced functioning, unemployment, financial stress, and interpersonal difficulties.
Usual onset	Often begins in childhood, however, onset is variable.
Duration	At least 6 months.
Causes	Heightened awareness of bodily sensations and the tendency to misinterpret bodily sensations.
Risk factors	Childhood neglect and abuse, chaotic lifestyle, history of substance and alcohol abuse, and psychosocial stressors.
Diagnostic method	Psychiatric assessment.
Differential diagnosis	Adjustment disorder, body dysmorphic disorder, obsessive-compulsive disorder, conversion disorder, and illness anxiety disorder.
Treatment	Cognitive-behavioral therapy, psychiatric medication, and brief psychodynamic interpersonal psychotherapy.
Medication	Selective serotonin reuptake inhibitor's and Serotonin–norepinephrine reuptake inhibitor's.
Prognosis	Often chronic but can be managed with the proper treatment.
Frequency	About 13-23% of the general population.

Somatic symptom disorder, also known as somatoform disorder, is defined by one or more chronic physical symptoms that coincide with excessive and maladaptive thoughts, emotions, and behaviors connected to those symptoms. The symptoms are not purposefully produced or feigned, and they may or may not coexist with a known medical ailment.

Manifestations of somatic symptom disorder are variable; symptoms can be widespread, specific, and often fluctuate. Somatic symptom disorder corresponds to the way an individual views and reacts to symptoms as rather than the symptoms themselves. Somatic symptom disorder may develop in those who suffer from an existing chronic illness or medical condition.

Several studies have found a high rate of comorbidity with major depressive disorder, generalized anxiety disorder, and phobias. Somatic symptom disorder is frequently associated with functional pain syndromes like fibromyalgia and IBS. Somatic symptom disorder typically leads to poor functioning, interpersonal issues, unemployment or problems at work, and financial strain as a result of excessive health-care visits.

The cause of somatic symptom disorder is unknown; however, somatic symptoms may result from a heightened awareness of specific physical sensations paired with a tendency to interpret these experiences as signs of a medical ailment.

Signs and Symptoms

Somatic symptom disorder can be detected by an ambiguous and often inconsistent history of symptoms that are rarely relieved by medical treatments. Additional signs of Somatic Symptom Disorder include interpreting normal sensations for medical ailments, avoiding physical activity, being disproportionately sensitive to medication side effects, and seeking medical care from several physicians for the same concerns.

Manifestations of Somatic symptom disorder are highly variable. Recurrent ailments usually begin before the age of 30; most patients have many somatic symptoms, while others only experience one. The severity may fluctuate, but symptoms rarely go away completely for long periods of time. Symptoms might be specific, such as regional pain and localized sensations, or general, such as fatigue, muscle aches, and malaise.

Those suffering from Somatic symptom disorder experience recurring and obsessive feelings and thoughts concerning their well-being. Common examples include severe anxiety regarding potential ailments, misinterpreting normal sensations as indications of severe illness, believing that symptoms are dangerous and serious despite lacking medical basis, claiming that medical evaluations and treatment have been inadequate, fearing that engaging in physical activity will harm the body, and spending a disproportionate amount of time thinking about symptoms.

Somatic symptoms disorder pertains to how an individual interprets and responds to symptoms as opposed to the symptoms themselves. Somatic symptom disorder can occur even in those who have an underlying chronic illness or medical condition. When a somatic symptom disorder coexists with another medical ailment, individuals overreact to the ailment's adverse effects. They may be unresponsive toward treatment or unusually sensitive to drug side effects. Those with somatic symptom disorder who also have another physical ailment may experience significant impairment that isn't expected from the condition.

Comorbidities

Most research that looked at additional mental illnesses or self-reported psychopathological symptoms among those with Somatic Symptom Disorder identified significant rates of comorbidity with depression and anxiety, but other psychiatric comorbidities were not usually looked at. Major depression, generalized anxiety disorder, and phobias were the most common concurrent conditions.

In studies evaluating different physical ailments, 41.5% of individuals with semantic dementia, 11.2% of subjects with Alzheimer's disease, 25% of female patients suffering from non-HIV lipodystrophy, and 18.5% of patients with congestive heart failure fulfilled Somatic symptom disorder criteria. 25.6% of individuals with fibromyalgia fulfilled Somatic symptom disorder criteria, and they exhibited higher depression rates than those with fibromyalgia who didn't meet the criteria for Somatic symptom disorder. In one study, 28.8% of those with Somatic Symptom Disorder had asthma, 23.1% had a heart condition, and 13.5% had gout, rheumatoid arthritis, or osteoarthritis.

Complications

Alcohol and drug abuse are frequently observed, and sometimes used to alleviate symptoms, increasing the risk of dependence on controlled substances. Other complications include poor functioning, problems with relationships, unemployment or difficulties at work, and financial stress due to excess health care visits.

Causes

Somatic symptoms can stem from a heightened awareness of sensations in the body, alongside the tendency to interpret those sensations as ailments. Studies suggest that risk factors of somatic symptoms include childhood neglect, sexual abuse, a chaotic lifestyle, and a history of substance and alcohol abuse. Psychosocial stressors, such as unemployment and reduced job performance, may also be risk factors. There could also be a genetic element. A study of monozygotic and dizygotic twins found that genetic components contributed 7% to 21% of somatic symptoms, with the remainder related to environmental factors. In another study, various single nucleotide polymorphisms were linked to somatic symptoms.

Psychological

Evidence suggests that along with more broad factors such as early childhood trauma or insecure attachment, negative psychological factors including catastrophizing, negative affectivity, rumination, avoidance, health anxiety, or a poor physical self-concept have a significant impact on the shift from unproblematic somatic symptoms to a severely debilitating somatic symptom disorder. Individuals who experience more negative psychological characteristics may regard medically unexplained symptoms to be more threatening and, therefore, exhibit stronger cognitive, emotional, and behavioral awareness of such symptoms. In addition, evidence suggests that negative psychological factors have a significant impact on the impairments and behaviors of people suffering from somatic symptom disorder, as well as the long-term stability of such symptoms.

Psychosocial

Psychosocial stresses and cultural norms influence how individuals present to their physicians. American and Korean individuals engaged in a study to measure somatization within the cultural context. It was discovered that Korean participants used more body-related phrases while discussing their connections with stressful events and experienced more sympathy when asked to read texts using somatic expressions when discussing their emotions.

Those raised in environments where expressing emotions during stages of development is discouraged face the highest risk of somatization. In primary care settings, studies indicated that somaticizing patients had much greater rates of unemployment and decreased occupational functioning than non-somaticizing patients.

Traumatic life events may cause the development of somatic symptom disorder. Most people with somatic symptom disorder originate from dysfunctional homes. A meta-analysis study revealed a connection between sexual abuse and functional gastrointestinal syndromes, chronic pain, non-epileptic seizures, and chronic pelvic pain.

Physiological

The hypothalamo pituitary adrenal axis (HPA) has a crucial role in stress response. While the HPA axis may become more active with depression, there is evidence of hypocortisolism in somatization. In somatic disorder, there is a negative connection between elevated pain scores and 5-hydroxy indol acetic acid (5-HIAA) and tryptophan levels.

It has been suggested that proinflammatory processes may have a role in somatic symptom disorder, such as an increase of non-specific somatic symptoms and sensitivity to painful stimuli. Proinflammatory activation and anterior cingulate cortex activity have been shown to be linked in those who experienced stressful life events for an extended period of time. It is further claimed that increased activity of the anterior cingulate cortex, which acts as a bridge between attention and emotion, leads to increased sensitivity of unwanted stimuli and bodily sensations.

Pain is a multifaceted experience, not just a sensation. While nociception refers to afferent neural activity that transmits sensory information in response to stimuli that may cause tissue damage, pain is a conscious experience requiring cortical activity and can occur in the absence of nociception. Those with somatic symptoms are thought to exaggerate their somatic symptoms through choice perception and perceive them in accordance with an ailment. This idea has been identified as a cognitive style known as "somatosensorial amplification." The term "central sensitization" has been created to describe the neurobiological notion that those predisposed to somatization have an overly sensitive neural network. Harmless and mild stimuli stimulate the nociceptive specific dorsal horn cells after central sensitization. As a result, pain is felt in response to stimuli that would not typically cause pain.

Genetic

Genetic investigations have suggested that genetic modifications connected to the monoaminergic system, in particular, may be relevant; nevertheless, a shared genetic source remains unknown. It is important to take into account the various processes involved in the development of somatic symptoms as well as the interactions between various biological and psychosocial factors. Given the high occurrence of trauma, particularly throughout childhood, it has been suggested that the epigenetic changes could be explanatory. Another study found that the glucocorticoid receptor gene (NR3C1) is hypomethylated in those with somatic symptom disorder and in those with depression.

Diagnosis

Because those with somatic syndrome disorder typically have a comprehensive previous workups, minimal laboratory testing is encouraged. Excessive testing increases the possibility of false-positive results, which may result in further interventions, associated risks, and greater expenses. While some practitioners order tests to reassure patients, research shows that diagnostic testing fails to alleviate somatic symptoms.

Specific tests, such as thyroid function assessments, urine drug screens, restricted blood studies, and minimal radiological imaging, may be conducted to rule out somatization because of medical issues.

Somatic Symptom Scale – 8

Main article: Somatic Symptom Scale - 8

The Somatic Symptom Scale – 8 (SSS-8) is a short self-report questionnaire that is used to evaluate somatic symptoms. It examines the perceived severity of common somatic symptoms. The SSS-8 is a condensed version of the well-known Patient Health Questionnaire-15 (PHQ-15).

On a five-point scale, respondents rate how much stomach or digestive issues, back discomfort, pain in the legs, arms, or joints, headaches, chest pain or shortness of breath, dizziness, feeling tired or having low energy, and trouble sleeping impacted them in the preceding seven days. Ratings are added together to provide a sum score that ranges from 0 to 32 points.

Diagnostic and Statistical Manual

The fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) modified the entry titled "somatoform disorders" to "somatic symptom and related disorders," in addition to modifying other diagnostic labels and criteria.

The DSM-5 criteria for Somatic Symptom Disorder includes "one or more somatic symptoms which are distressing or result in substantial impairment of daily life." Additional criteria, often known as B criteria, include "excessive thoughts, feelings, or behaviors regarding somatic symptoms or corresponding health concerns manifested by disproportionate and persistent thoughts about the severity of one's symptoms." According to the DSM, "Although any one somatic symptom might not be consistently present, one's state of being symptomatic is continuous (typically lasting more than 6 months)."

The DSM includes five distinct descriptions for Somatic Symptom Disorder. These include somatic symptom disorder with predominant pain, formally referred to as pain disorder, as well as classifications for mild, moderate, and severe symptoms.

International Classification of Diseases

The ICD-11 classifies somatic symptoms as "Bodily distress disorder". Bodily distress disorder is characterized by the presence of distressing bodily symptoms and excessive attention devoted to those symptoms. The ICD-11 further specifies that if another health condition is causing or contributing to the symptoms, the level of attention must be clearly excessive in relation to the nature and course of the condition.

Differential diagnosis

Somatic syndrome disorder's widespread, non-specific symptoms may conceal and mimic the manifestations of other medical disorders, making diagnosis and therapy challenging. Adjustment disorder, body dysmorphic disorder, obsessive-compulsive disorder, and illness anxiety disorder may all exhibit excessive and exaggerated emotional and behavioral responses. Other functional diseases with unknown etiology, such as fibromyalgia and irritable bowel syndrome, tend not to present with excessive thoughts, feelings, or maladaptive behavior.

Somatic symptom disorder overlaps with illness anxiety disorder and conversion disorder. Illness anxiety disorder is characterized by an obsession with having or developing a dangerous, undetected medical ailment, despite the absence of bodily symptoms. Conversion disorder may present with one or more symptoms of various sorts. Motor symptoms involve weakness or paralysis; aberrant movements including tremor or dystonic movements; abnormal gait patterns; and abnormal limb posture. The presenting symptom in conversion disorder is loss of function, but in somatic symptom disorder, the emphasis is on the discomfort that specific symptoms produce. Conversion disorder often lacks the overwhelming thoughts, feelings, and behaviors that characterize somatic symptom disorder.

Treatment

Instead than focusing on treating the symptoms, the key objective is to support the patient in coping with physical symptoms such as health anxiety and harmful behaviors.

Early psychiatric treatment is advised. Evidence suggests that SSRIs and SNRIs can lower pain perception. However, because individuals with Somatic Symptom Disorder may have a low threshold for experiencing adverse reactions, medications should be started at the lowest possible dose and gradually increased to produce a therapeutic effect.

Cognitive-behavioral therapy has been linked to significant improvements in patient-reported function and somatic symptoms, a reduction in health-care expenses, and a reduction in symptoms of depression. Furthermore, brief psychodynamic interpersonal psychotherapy (PIT) for patients with Somatic Symptom Disorder has been proven to improve the physical quality of life in patients with many, difficult-to-treat, medically unexplained symptoms over time.

CBT can help in some of the following ways:

• Learn to reduce stress
• Learn to cope with physical symptoms
• Learn to deal with depression and other psychological issues
• Improve quality of life
• Reduce preoccupation with symptom

Outlook

Somatic symptom disorder is typically persistent, with symptoms that wax and wane. Chronic limitations in general function, substantial psychological impairment, and a reduction in quality of life are all common. Some investigations, however, have found that individuals can recover; the natural history of the illnesses implies that around 50% to 75% of patients with medically unexplained symptoms improve, whereas 10% to 30% deteriorate. Fewer physical symptoms and better baseline functioning are stronger prognostic indicators. A strong, positive relationship between the physician and the patient is crucial, and it should be accompanied by frequent, supportive visits to avoid the temptation to medicate or test when these interventions are not obviously necessary.

Epidemiology

Somatic symptom disorder affects 5% to 7% of the general population, with a higher female representation, and can arise throughout childhood, adolescence, or adulthood. Evidence suggests that the emergence of prodromal symptoms often begins in childhood and that symptoms fitting the criteria for somatic symptom disorder are common during adolescence. A community study of adolescents found that 5% had persistent distressing physical symptoms paired with psychological concerns. In the primary care patient population, the rate rises to around 17%. Patients with functional illnesses such as fibromyalgia, irritable bowel syndrome, and chronic fatigue syndrome have a greater prevalence of somatic symptom disorder. The reported frequency of somatic symptom disorder, as defined by DSM-5 criteria, ranges from 25 to 60% among these patients.

History

Somatization is an idea that physicians have been attempting to comprehend since the dawn of time. The Egyptians and Sumerians were reported to have utilized the notions of melancholia and hysteria as early as 2600 BC. For many years, somatization was used in conjunction with the terms hysteria, melancholia, and hypochondriasis.

During the 17th century, knowledge of the central nervous system grew, giving rise to the notion that numerous inexplicable illnesses could be linked to the brain. Thomas Willis, widely regarded as the father of neurology, recognized hysteria in women and hypochondria in males as brain disorders. Thomas Sydenham contributed significantly to the belief that hysteria and hypochondria are mental rather than physical illnesses. The term "English Malady" was used by George Cheyne to denote that hysteria and hypochondriasis are brain and/or mind-related disorders.

Wilhelm Stekel, a German psychoanalyst, was the first to introduce the term somatization, and Paul Briquet was the first to characterize what is now known as Somatic symptom disorder. Briquet reported individuals who had been unwell for most of their lives and complained of a variety of symptoms from various organ systems. Despite many appointments, hospitalizations, and tests, symptoms continue. Somatization disorder was later dubbed "Briquet Syndrome" in his honor. Over time, the concept of hysteria was used in place of a personality or character type, conversion responses, phobia, and anxiety to accompany psychoneuroses, and its incorporation in everyday English as a negative word led to a distancing from this concept.

Controversy

Somatic symptom disorder has long been a contentious diagnosis because it was based solely on negative criteria, namely the absence of a medical explanation for the presenting physical problems. As a result, any person suffering from a poorly understood illness may meet the criteria for this psychological diagnosis, regardless of whether they exhibit psychiatric symptoms in the traditional sense.

Misdiagnosis

In the opinion of Allen Frances, chair of the DSM-IV task force, the DSM-5's somatic symptom disorder brings with it a risk of mislabeling a sizable proportion of the population as mentally ill.

Millions of people could be mislabeled, with the burden falling disproportionately on women, because they are more likely to be casually dismissed as 'catastrophizers' when presenting with physical symptoms.

Trans-splicing

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Trans-splicing 

Trans-splicing is a special form of RNA processing where exons from two different primary RNA transcripts are joined end to end and ligated. It is usually found in eukaryotes and mediated by the spliceosome, although some bacteria and archaea also have "half-genes" for tRNAs.

Genic trans-splicing

Whereas "normal" (cis-)splicing processes a single molecule, trans-splicing generates a single RNA transcript from multiple separate pre-mRNAs. This phenomenon can be exploited for molecular therapy to address mutated gene products. Genic trans-splicing allows variability in RNA diversity and increases proteome complexity.

Oncogenesis

While some fusion transcripts occur via trans-splicing in normal human cells, trans-splicing can also be the mechanism behind certain oncogenic fusion transcripts.

SL trans-splicing

Spliced leader (SL) trans-splicing is used by certain microorganisms, notably protists of the Kinetoplastea class to express genes. In these organisms, a capped splice leader RNA is transcribed, and simultaneously, genes are transcribed in long polycistrons. The capped splice leader is trans-spliced onto each gene to generate monocistronic capped and polyadenylated transcripts. These early-diverging eukaryotes use few introns, and the spliceosome they possess show some unusual variations in their structure assembly. They also possess multiple eIF4E isoforms with specialized roles in capping. The spliced leader sequence is highly conserved in lower species that undergo trans-splicing. Such as trypanosomes. While the spliced leader's role is not known in the cell, it's thought to be involved in translation initiation. In C.elegans, the splicing of the sequence leader occurs close to the initiation codon. Some scientists also suggest the sequence is required for cell viability. In Ascaris, the spliced leader sequence is needed to the RNA gene can be transcribed. The Spliced leader sequence may be responsible for initiation, mRNA localization, and translation initiation or inhibition.

Some other eukaryotes, notably among dinoflagellates, sponges, nematodes, cnidarians, ctenophores, flatworms, crustaceans, chaetognaths, rotifers, and tunicates also use more or less frequently the SL trans-splicing. In the tunicate Ciona intestinalis, the extent of SL trans-splicing is better described by a quantitative view recognising frequently and infrequently trans-spliced genes rather than a binary and conventional categorisation of trans-spliced versus non-trans-spliced genes.

The SL trans-splicing functions in the resolution of polycistronic transcripts of operons into individual 5'-capped mRNAs. This processing is achieved when the outrons are trans-spliced to unpaired, downstream acceptor sites adjacent to cistron open reading frames.

Mechanism

Trans-splicing is characterized by the joining of two separate exons transcribed RNAs. The signal for this splicing is the outron at the 5’ end of the mRNA, in the absence of a functional 5’ splice site upstream. When the 5’ outron in spliced, the 5’ splice site of the spliced leader RNA is branched to the outron and forms an intermediate. This step results in a free spliced leader exon. The exon is then spliced to the first exon on the pre-mRNA and the intermediate is released. Trans-splicing differs from cis-splicing in that there is no 5' splice site on the pre-mRNA. Instead the 5' splice site is provided by the SL sequence.

Trans-splicing between sense and anti-sense strands

As a result of the sense strand undergoing transcription, a pre-mRNA is formed that complements the sense strand. The anti-sense strand is also transcribed resulting in a complementary pre-mRNA strand. The exons from the two transcripts are spliced together to form a chimeric mRNA.

Alternative Trans-splicing

Alternative trans-splicing includes intragenic trans-splicing and intergenic trans-splicing. Intragenic trans-splicing involves duplication of exons in the pre-mRNA. Intergenic trans-splicing is characterized by the splicing together of exons formed form the pre-mRNA of two different genes, resulting in trans-genic mRNA.

Alternative splicing

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Alternative_splicing 

Alternative splicing produces three protein isoforms. Protein A includes all of the exons, whereas Proteins B and C result from exon skipping.

Alternative splicing, or alternative RNA splicing, or differential splicing, is an alternative splicing process during gene expression that allows a single gene to code for multiple proteins. In this process, particular exons of a gene may be included within or excluded from the final, processed messenger RNA (mRNA) produced from that gene. This means the exons are joined in different combinations, leading to different (alternative) mRNA strands. Consequently, the proteins translated from alternatively spliced mRNAs usually contain differences in their amino acid sequence and, often, in their biological functions (see Figure).

Biologically relevant alternative splicing occurs as a normal phenomenon in eukaryotes, where it increases the number of proteins that can be encoded by the genome. In humans, it is widely believed that ~95% of multi-exonic genes are alternatively spliced to produce functional alternative products from the same gene but many scientists believe that most of the observed splice variants are due to splicing errors and the actual number of biologically relevant alternatively spliced genes is much lower.

Alternative splicing enables the regulated generation of multiple mRNA and protein products from a single gene.

There are numerous modes of alternative splicing observed, of which the most common is exon skipping. In this mode, a particular exon may be included in mRNAs under some conditions or in particular tissues, and omitted from the mRNA in others.

The production of alternatively spliced mRNAs is regulated by a system of trans-acting proteins that bind to cis-acting sites on the primary transcript itself. Such proteins include splicing activators that promote the usage of a particular splice site, and splicing repressors that reduce the usage of a particular site. Mechanisms of alternative splicing are highly variable, and new examples are constantly being found, particularly through the use of high-throughput techniques. Researchers hope to fully elucidate the regulatory systems involved in splicing, so that alternative splicing products from a given gene under particular conditions ("splicing variants") could be predicted by a "splicing code".

Abnormal variations in splicing are also implicated in disease; a large proportion of human genetic disorders result from splicing variants. Abnormal splicing variants are also thought to contribute to the development of cancer, and splicing factor genes are frequently mutated in different types of cancer.

Discovery

Alternative splicing was first observed in 1977. The adenovirus produces five primary transcripts early in its infectious cycle, prior to viral DNA replication, and an additional one later, after DNA replication begins. The early primary transcripts continue to be produced after DNA replication begins. The additional primary transcript produced late in infection is large and comes from 5/6 of the 32kb adenovirus genome. This is much larger than any of the individual adenovirus mRNAs present in infected cells. Researchers found that the primary RNA transcript produced by adenovirus type 2 in the late phase was spliced in many different ways, resulting in mRNAs encoding different viral proteins. In addition, the primary transcript contained multiple polyadenylation sites, giving different 3’ ends for the processed mRNAs.

In 1981, the first example of alternative splicing in a transcript from a normal, endogenous gene was characterized. The gene encoding the thyroid hormone calcitonin was found to be alternatively spliced in mammalian cells. The primary transcript from this gene contains 6 exons; the calcitonin mRNA contains exons 1–4, and terminates after a polyadenylation site in exon 4. Another mRNA is produced from this pre-mRNA by skipping exon 4, and includes exons 1–3, 5, and 6. It encodes a protein known as CGRP (calcitonin gene related peptide). Examples of alternative splicing in immunoglobin gene transcripts in mammals were also observed in the early 1980s.

Since then, many other examples of biologically relevant alternative splicing have been found in eukaryotes. The "record-holder" for alternative splicing is a D. melanogaster gene called Dscam, which could potentially have 38,016 splice variants.

In 2021, it was discovered that the genome of adenovirus type 2, the adenovirus in which alternative splicing was first identified, was able to produce a much greater variety of mRNA than previously thought. By using next generation sequencing technology, researchers were able to update the human adenovirus type 2 transcriptome, and present a mind-boggling 904 unique mRNA, produced by the virus through a complex pattern of alternative splicing. Very few of these splice variants have been shown to be functional, a point that the authors raise in their paper.

"An outstanding question is what roles the menagerie of novel RNAs play or whether they are spurious molecules generated by an overloaded splicing machinery."

Modes

Traditional classification of basic types of alternative RNA splicing events. Exons are represented as blue and yellow blocks, introns as lines in between.

Relative frequencies of types of alternative splicing events differ between humans and fruit flies.

Five basic modes of alternative splicing are generally recognized.

• Exon skipping or cassette exon: in this case, an exon may be spliced out of the primary transcript or retained. This is the most common mode in mammalian pre-mRNAs.
• Mutually exclusive exons: One of two exons is retained in mRNAs after splicing, but not both.
• Alternative donor site: An alternative 5' splice junction (donor site) is used, changing the 3' boundary of the upstream exon.
• Alternative acceptor site: An alternative 3' splice junction (acceptor site) is used, changing the 5' boundary of the downstream exon.
• Intron retention: A sequence may be spliced out as an intron or simply retained. This is distinguished from exon skipping because the retained sequence is not flanked by introns. If the retained intron is in the coding region, the intron must encode amino acids in frame with the neighboring exons, or a stop codon or a shift in the reading frame will cause the protein to be non-functional. This is the rarest mode in mammals but the most common in plants.

In addition to these primary modes of alternative splicing, there are two other main mechanisms by which different mRNAs may be generated from the same gene; multiple promoters and multiple polyadenylation sites. Use of multiple promoters is properly described as a transcriptional regulation mechanism rather than alternative splicing; by starting transcription at different points, transcripts with different 5'-most exons can be generated. At the other end, multiple polyadenylation sites provide different 3' end points for the transcript. Both of these mechanisms are found in combination with alternative splicing and provide additional variety in mRNAs derived from a gene.

Schematic cutoff from 3 splicing structures in the murine hyaluronidase gene. Directionality of transcription from 5' to 3' is shown from left to right. Exons and introns are not drawn to scale.

These modes describe basic splicing mechanisms, but may be inadequate to describe complex splicing events. For instance, the figure to the right shows 3 spliceforms from the mouse hyaluronidase 3 gene. Comparing the exonic structure shown in the first line (green) with the one in the second line (yellow) shows intron retention, whereas the comparison between the second and the third spliceform (yellow vs. blue) exhibits exon skipping. A model nomenclature to uniquely designate all possible splicing patterns has recently been proposed.

Mechanisms

General splicing mechanism

Main article: RNA splicing

Spliceosome A complex defines the 5' and 3' ends of the intron before removal

When the pre-mRNA has been transcribed from the DNA, it includes several introns and exons. (In nematodes, the mean is 4–5 exons and introns; in the fruit fly Drosophila there can be more than 100 introns and exons in one transcribed pre-mRNA.) The exons to be retained in the mRNA are determined during the splicing process. The regulation and selection of splice sites are done by trans-acting splicing activator and splicing repressor proteins as well as cis-acting elements within the pre-mRNA itself such as exonic splicing enhancers and exonic splicing silencers.

The typical eukaryotic nuclear intron has consensus sequences defining important regions. Each intron has the sequence GU at its 5' end. Near the 3' end there is a branch site. The nucleotide at the branchpoint is always an A; the consensus around this sequence varies somewhat. In humans the branch site consensus sequence is yUnAy. The branch site is followed by a series of pyrimidines – the polypyrimidine tract – then by AG at the 3' end.

Splicing of mRNA is performed by an RNA and protein complex known as the spliceosome, containing snRNPs designated U1, U2, U4, U5, and U6 (U3 is not involved in mRNA splicing). U1 binds to the 5' GU and U2, with the assistance of the U2AF protein factors, binds to the branchpoint A within the branch site. The complex at this stage is known as the spliceosome A complex. Formation of the A complex is usually the key step in determining the ends of the intron to be spliced out, and defining the ends of the exon to be retained. (The U nomenclature derives from their high uridine content).

The U4,U5,U6 complex binds, and U6 replaces the U1 position. U1 and U4 leave. The remaining complex then performs two transesterification reactions. In the first transesterification, 5' end of the intron is cleaved from the upstream exon and joined to the branch site A by a 2',5'-phosphodiester linkage. In the second transesterification, the 3' end of the intron is cleaved from the downstream exon, and the two exons are joined by a phosphodiester bond. The intron is then released in lariat form and degraded.

Regulatory elements and proteins

Splicing repression

Splicing is regulated by trans-acting proteins (repressors and activators) and corresponding cis-acting regulatory sites (silencers and enhancers) on the pre-mRNA. However, as part of the complexity of alternative splicing, it is noted that the effects of a splicing factor are frequently position-dependent. That is, a splicing factor that serves as a splicing activator when bound to an intronic enhancer element may serve as a repressor when bound to its splicing element in the context of an exon, and vice versa. The secondary structure of the pre-mRNA transcript also plays a role in regulating splicing, such as by bringing together splicing elements or by masking a sequence that would otherwise serve as a binding element for a splicing factor. Together, these elements form a "splicing code" that governs how splicing will occur under different cellular conditions.

There are two major types of cis-acting RNA sequence elements present in pre-mRNAs and they have corresponding trans-acting RNA-binding proteins. Splicing silencers are sites to which splicing repressor proteins bind, reducing the probability that a nearby site will be used as a splice junction. These can be located in the intron itself (intronic splicing silencers, ISS) or in a neighboring exon (exonic splicing silencers, ESS). They vary in sequence, as well as in the types of proteins that bind to them. The majority of splicing repressors are heterogeneous nuclear ribonucleoproteins (hnRNPs) such as hnRNPA1 and polypyrimidine tract binding protein (PTB). Splicing enhancers are sites to which splicing activator proteins bind, increasing the probability that a nearby site will be used as a splice junction. These also may occur in the intron (intronic splicing enhancers, ISE) or exon (exonic splicing enhancers, ESE). Most of the activator proteins that bind to ISEs and ESEs are members of the SR protein family. Such proteins contain RNA recognition motifs and arginine and serine-rich (RS) domains.

Splicing activation

In general, the determinants of splicing work in an inter-dependent manner that depends on context, so that the rules governing how splicing is regulated form a splicing code. The presence of a particular cis-acting RNA sequence element may increase the probability that a nearby site will be spliced in some cases, but decrease the probability in other cases, depending on context. The context within which regulatory elements act includes cis-acting context that is established by the presence of other RNA sequence features, and trans-acting context that is established by cellular conditions. For example, some cis-acting RNA sequence elements influence splicing only if multiple elements are present in the same region so as to establish context. As another example, a cis-acting element can have opposite effects on splicing, depending on which proteins are expressed in the cell (e.g., neuronal versus non-neuronal PTB). The adaptive significance of splicing silencers and enhancers is attested by studies showing that there is strong selection in human genes against mutations that produce new silencers or disrupt existing enhancers.

DNA methylation and alternative splicing in social insects

CpG DNA methylation has showed a role to regulate the alternative splicing in social insects. In honey bees (Apis mellifera), CpG DNA methylation seems to regulate the exon skipping based on the first few genomic studies after honey bee genome was available. CpG DNA methylation regulated alternative splicing more extensively, not only affect exon skipping, but also intron retention, and other splicing events.

Examples

Exon skipping: Drosophila dsx

Alternative splicing of dsx pre-mRNA

Pre-mRNAs from the D. melanogaster gene dsx contain 6 exons. In males, exons 1,2,3,5,and 6 are joined to form the mRNA, which encodes a transcriptional regulatory protein required for male development. In females, exons 1,2,3, and 4 are joined, and a polyadenylation signal in exon 4 causes cleavage of the mRNA at that point. The resulting mRNA is a transcriptional regulatory protein required for female development.

This is an example of exon skipping. The intron upstream from exon 4 has a polypyrimidine tract that doesn't match the consensus sequence well, so that U2AF proteins bind poorly to it without assistance from splicing activators. This 3' splice acceptor site is therefore not used in males. Females, however, produce the splicing activator Transformer (Tra) (see below). The SR protein Tra2 is produced in both sexes and binds to an ESE in exon 4; if Tra is present, it binds to Tra2 and, along with another SR protein, forms a complex that assists U2AF proteins in binding to the weak polypyrimidine tract. U2 is recruited to the associated branchpoint, and this leads to inclusion of exon 4 in the mRNA.

Alternative acceptor sites: Drosophila Transformer

Alternative splicing of the Drosophila Transformer gene product.

Pre-mRNAs of the Transformer (Tra) gene of Drosophila melanogaster undergo alternative splicing via the alternative acceptor site mode. The gene Tra encodes a protein that is expressed only in females. The primary transcript of this gene contains an intron with two possible acceptor sites. In males, the upstream acceptor site is used. This causes a longer version of exon 2 to be included in the processed transcript, including an early stop codon. The resulting mRNA encodes a truncated protein product that is inactive. Females produce the master sex determination protein Sex lethal (Sxl). The Sxl protein is a splicing repressor that binds to an ISS in the RNA of the Tra transcript near the upstream acceptor site, preventing U2AF protein from binding to the polypyrimidine tract. This prevents the use of this junction, shifting the spliceosome binding to the downstream acceptor site. Splicing at this point bypasses the stop codon, which is excised as part of the intron. The resulting mRNA encodes an active Tra protein, which itself is a regulator of alternative splicing of other sex-related genes (see dsx above).

Exon definition: Fas receptor

Alternative splicing of the Fas receptor pre-mRNA

Multiple isoforms of the Fas receptor protein are produced by alternative splicing. Two normally occurring isoforms in humans are produced by an exon-skipping mechanism. An mRNA including exon 6 encodes the membrane-bound form of the Fas receptor, which promotes apoptosis, or programmed cell death. Increased expression of Fas receptor in skin cells chronically exposed to the sun, and absence of expression in skin cancer cells, suggests that this mechanism may be important in elimination of pre-cancerous cells in humans. If exon 6 is skipped, the resulting mRNA encodes a soluble Fas protein that does not promote apoptosis. The inclusion or skipping of the exon depends on two antagonistic proteins, TIA-1 and polypyrimidine tract-binding protein (PTB).

• The 5' donor site in the intron downstream from exon 6 in the pre-mRNA has a weak agreement with the consensus sequence, and is not bound usually by the U1 snRNP. If U1 does not bind, the exon is skipped (see "a" in accompanying figure).
• Binding of TIA-1 protein to an intronic splicing enhancer site stabilizes binding of the U1 snRNP. The resulting 5' donor site complex assists in binding of the splicing factor U2AF to the 3' splice site upstream of the exon, through a mechanism that is not yet known.
• Exon 6 contains a pyrimidine-rich exonic splicing silencer, ure6, where PTB can bind. If PTB binds, it inhibits the effect of the 5' donor complex on the binding of U2AF to the acceptor site, resulting in exon skipping.

This mechanism is an example of exon definition in splicing. A spliceosome assembles on an intron, and the snRNP subunits fold the RNA so that the 5' and 3' ends of the intron are joined. However, recently studied examples such as this one show that there are also interactions between the ends of the exon. In this particular case, these exon definition interactions are necessary to allow the binding of core splicing factors prior to assembly of the spliceosomes on the two flanking introns.

Repressor-activator competition: HIV-1 tat exon 2

Alternative splicing of HIV-1 tat exon 2

HIV, the retrovirus that causes AIDS in humans, produces a single primary RNA transcript, which is alternatively spliced in multiple ways to produce over 40 different mRNAs. Equilibrium among differentially spliced transcripts provides multiple mRNAs encoding different products that are required for viral multiplication. One of the differentially spliced transcripts contains the tat gene, in which exon 2 is a cassette exon that may be skipped or included. The inclusion of tat exon 2 in the RNA is regulated by competition between the splicing repressor hnRNP A1 and the SR protein SC35. Within exon 2 an exonic splicing silencer sequence (ESS) and an exonic splicing enhancer sequence (ESE) overlap. If A1 repressor protein binds to the ESS, it initiates cooperative binding of multiple A1 molecules, extending into the 5’ donor site upstream of exon 2 and preventing the binding of the core splicing factor U2AF35 to the polypyrimidine tract. If SC35 binds to the ESE, it prevents A1 binding and maintains the 5’ donor site in an accessible state for assembly of the spliceosome. Competition between the activator and repressor ensures that both mRNA types (with and without exon 2) are produced.

Adaptive significance

Genuine alternative splicing occurs in both protein-coding genes and non-coding genes to produce multiple products (proteins or non-coding RNAs). External information is needed in order to decide which product is made, given a DNA sequence and the initial transcript. Since the methods of regulation are inherited, this provides novel ways for mutations to affect gene expression.

Alternative splicing may provide evolutionary flexibility. A single point mutation may cause a given exon to be occasionally excluded or included from a transcript during splicing, allowing production of a new protein isoform without loss of the original protein. Studies have identified intrinsically disordered regions (see Intrinsically unstructured proteins) as enriched in the non-constitutive exons suggesting that protein isoforms may display functional diversity due to the alteration of functional modules within these regions. Such functional diversity achieved by isoforms is reflected by their expression patterns and can be predicted by machine learning approaches. Comparative studies indicate that alternative splicing preceded multicellularity in evolution, and suggest that this mechanism might have been co-opted to assist in the development of multicellular organisms.

Research based on the Human Genome Project and other genome sequencing has shown that humans have only about 30% more genes than the roundworm Caenorhabditis elegans, and only about twice as many as the fly Drosophila melanogaster. This finding led to speculation that the perceived greater complexity of humans, or vertebrates generally, might be due to higher rates of alternative splicing in humans than are found in invertebrates. However, a study on samples of 100,000 expressed sequence tags (EST) each from human, mouse, rat, cow, fly (D. melanogaster), worm (C. elegans), and the plant Arabidopsis thaliana found no large differences in frequency of alternatively spliced genes among humans and any of the other animals tested. Another study, however, proposed that these results were an artifact of the different numbers of ESTs available for the various organisms. When they compared alternative splicing frequencies in random subsets of genes from each organism, the authors concluded that vertebrates do have higher rates of alternative splicing than invertebrates.

Disease

Changes in the RNA processing machinery may lead to mis-splicing of multiple transcripts, while single-nucleotide alterations in splice sites or cis-acting splicing regulatory sites may lead to differences in splicing of a single gene, and thus in the mRNA produced from a mutant gene's transcripts. A study in 2005 involving probabilistic analyses indicated that greater than 60% of human disease-causing mutations affect splicing rather than directly affecting coding sequences. A more recent study indicates that one-third of all hereditary diseases are likely to have a splicing component. Regardless of exact percentage, a number of splicing-related diseases do exist. As described below, a prominent example of splicing-related diseases is cancer.

Abnormally spliced mRNAs are also found in a high proportion of cancerous cells. Combined RNA-Seq and proteomics analyses have revealed striking differential expression of splice isoforms of key proteins in important cancer pathways. It is not always clear whether such aberrant patterns of splicing contribute to the cancerous growth, or are merely consequence of cellular abnormalities associated with cancer. For certain types of cancer, like in colorectal and prostate, the number of splicing errors per cancer has been shown to vary greatly between individual cancers, a phenomenon referred to as transcriptome instability. Transcriptome instability has further been shown to correlate grealty with reduced expression level of splicing factor genes. Mutation of DNMT3A has been demonstrated to contribute to hematologic malignancies, and that DNMT3A-mutated cell lines exhibit transcriptome instability as compared to their isogenic wildtype counterparts.

In fact, there is actually a reduction of alternative splicing in cancerous cells compared to normal ones, and the types of splicing differ; for instance, cancerous cells show higher levels of intron retention than normal cells, but lower levels of exon skipping. Some of the differences in splicing in cancerous cells may be due to the high frequency of somatic mutations in splicing factor genes, and some may result from changes in phosphorylation of trans-acting splicing factors. Others may be produced by changes in the relative amounts of splicing factors produced; for instance, breast cancer cells have been shown to have increased levels of the splicing factor SF2/ASF. One study found that a relatively small percentage (383 out of over 26000) of alternative splicing variants were significantly higher in frequency in tumor cells than normal cells, suggesting that there is a limited set of genes which, when mis-spliced, contribute to tumor development. It is believed however that the deleterious effects of mis-spliced transcripts are usually safeguarded and eliminated by a cellular posttranscriptional quality control mechanism termed nonsense-mediated mRNA decay [NMD].

One example of a specific splicing variant associated with cancers is in one of the human DNMT genes. Three DNMT genes encode enzymes that add methyl groups to DNA, a modification that often has regulatory effects. Several abnormally spliced DNMT3B mRNAs are found in tumors and cancer cell lines. In two separate studies, expression of two of these abnormally spliced mRNAs in mammalian cells caused changes in the DNA methylation patterns in those cells. Cells with one of the abnormal mRNAs also grew twice as fast as control cells, indicating a direct contribution to tumor development by this product.

Another example is the Ron (MST1R) proto-oncogene. An important property of cancerous cells is their ability to move and invade normal tissue. Production of an abnormally spliced transcript of Ron has been found to be associated with increased levels of the SF2/ASF in breast cancer cells. The abnormal isoform of the Ron protein encoded by this mRNA leads to cell motility.

Overexpression of a truncated splice variant of the FOSB gene – ΔFosB – in a specific population of neurons in the nucleus accumbens has been identified as the causal mechanism involved in the induction and maintenance of an addiction to drugs and natural rewards.

Recent provocative studies point to a key function of chromatin structure and histone modifications in alternative splicing regulation. These insights suggest that epigenetic regulation determines not only what parts of the genome are expressed but also how they are spliced.

Genome-wide analysis

Genome-wide analysis of alternative splicing is a challenging task. Typically, alternatively spliced transcripts have been found by comparing EST sequences, but this requires sequencing of very large numbers of ESTs. Most EST libraries come from a very limited number of tissues, so tissue-specific splice variants are likely to be missed in any case. High-throughput approaches to investigate splicing have, however, been developed, such as: DNA microarray-based analyses, RNA-binding assays, and deep sequencing. These methods can be used to screen for polymorphisms or mutations in or around splicing elements that affect protein binding. When combined with splicing assays, including in vivo reporter gene assays, the functional effects of polymorphisms or mutations on the splicing of pre-mRNA transcripts can then be analyzed.

In microarray analysis, arrays of DNA fragments representing individual exons (e.g. Affymetrix exon microarray) or exon/exon boundaries (e.g. arrays from ExonHit or Jivan) have been used. The array is then probed with labeled cDNA from tissues of interest. The probe cDNAs bind to DNA from the exons that are included in mRNAs in their tissue of origin, or to DNA from the boundary where two exons have been joined. This can reveal the presence of particular alternatively spliced mRNAs.

CLIP (Cross-linking and immunoprecipitation) uses UV radiation to link proteins to RNA molecules in a tissue during splicing. A trans-acting splicing regulatory protein of interest is then precipitated using specific antibodies. When the RNA attached to that protein is isolated and cloned, it reveals the target sequences for that protein. Another method for identifying RNA-binding proteins and mapping their binding to pre-mRNA transcripts is "Microarray Evaluation of Genomic Aptamers by shift (MEGAshift)".net This method involves an adaptation of the "Systematic Evolution of Ligands by Exponential Enrichment (SELEX)" method together with a microarray-based readout. Use of the MEGAshift method has provided insights into the regulation of alternative splicing by allowing for the identification of sequences in pre-mRNA transcripts surrounding alternatively spliced exons that mediate binding to different splicing factors, such as ASF/SF2 and PTB. This approach has also been used to aid in determining the relationship between RNA secondary structure and the binding of splicing factors.

Deep sequencing technologies have been used to conduct genome-wide analyses of mRNAs – unprocessed and processed – thus providing insights into alternative splicing. For example, results from use of deep sequencing indicate that, in humans, an estimated 95% of transcripts from multiexon genes undergo alternative splicing, with a number of pre-mRNA transcripts spliced in a tissue-specific manner. Functional genomics and computational approaches based on multiple instance learning have also been developed to integrate RNA-seq data to predict functions for alternatively spliced isoforms. Deep sequencing has also aided in the in vivo detection of the transient lariats that are released during splicing, the determination of branch site sequences, and the large-scale mapping of branchpoints in human pre-mRNA transcripts.

Use of reporter assays makes it possible to find the splicing proteins involved in a specific alternative splicing event by constructing reporter genes that will express one of two different fluorescent proteins depending on the splicing reaction that occurs. This method has been used to isolate mutants affecting splicing and thus to identify novel splicing regulatory proteins inactivated in those mutants.

Recent advancements in protein structure prediction have facilitated the development of new tools for genome annotation and alternative splicing anlaysis. For instance, isoform.io, a platform guided by protein structure predictions, has evaluated hundreds of thousands of isoforms of human protein-coding genes assembled from numerous RNA sequencing experiments across a variety of human tissues. This comprehensive analysis has led to the identification of numerous isoforms with more confidently predicted structure and potentially superior function compared to canonical isoforms in the latest human gene database. By integrating structural predictions with expression and evolutionary evidence, this approach has demonstrated the potential of protein structure prediction as a tool for refining the annotation of the human genome.

Databases

There is a collection of alternative splicing databases. These databases are useful for finding genes having pre-mRNAs undergoing alternative splicing and alternative splicing events or to study the functional impact of alternative splicing.

• AspicDB database
• Intronerator database
• ProSAS database