Principles of Molecular Diseases PDF

The Principles of Molecular Diseases 1. The effects of variations 85% of the variations are found in the coding sequence. The great majority of these variations cause a loss of function, but also some gain of functions or novel properties (alteration of the protein function) are seen. The...

The Principles of Molecular Diseases 1. The effects of variations 85% of the variations are found in the coding sequence. The great majority of these variations cause a loss of function, but also some gain of functions or novel properties (alteration of the protein function) are seen. The mechanisms underlying the mutations are the following: - stability of RNA – altered protein amount. - splicing mechanisms which result in abnormal proteins. - regulation of genes – variation does not affect coding sequence, but the variation affects the regulatory elements or the dosage of these genes. In this case you can expect increased or decreased amounts of normal proteins. There are some scenarios in which the variation, which is in the regulatory element, results in ectopic or heterochronic expression of this protein. Especially variation in the regulatory elements, regulating the genes involved in development results in an expression in the wrong place at the wrong time. 2. Number of genes are involved by single variation Single function mutation: In mendelian conditions only a single gene is involved, such as Thalassemia, Cystic Fibrosis, Sickle anemia and Phenylketonuria. Multiple function mutation: the same variation affects multiple genes directly or indirectly: - Directly: Large scale mutations such as chromosomal aberrations or contiguous genes syndrome (ex. WBS). - Indirectly: Simple mutation in a regulatory gene such as SMS (RAI1), CARGE (CHD7), ATRX, RETT (MECP2), Fragile X syndrome (FMR1). 3. Effect of a pathogenic variant on a gene function Loss of function – decreased level of expression of protein. This can be due to a mutation that interrupts the gene so that there is a complete expression failure or a reduction in the expression. In case of reduction of expression of protein it is called hypomorphic polymorphism. Gain of function – are heterogeneous variations, because they cause increased expression level, this can be due to an increased expression of the protein or to an activating point mutation in the promoter. Altered function (New function). 4. Nomenclature for describing the effect of an allele Null allele or amorph: an allele that produces no product. Hypomorph: an allele that produces reduced amount or activity of product. Hypermorph: an allele that produces increased amount or activity of product. Neomorph: an allele with a novel activity or product. Antimorph: an allele whose activity or product antagonizes the activity of the normal product. 5. Loss of function without dosage sensitive genes Loss of function – also consider whether the gene involved is dosage sensitive or non-dosage sensitive. Not dosage sensitive genes show a recessive behaviour (green line), 2 mutations in both alleles are needed. 1 The pathogenesis is only shown when the amount of product is very very low such as 5%, 10 % or not at all. Congenital Adrenal Hyperplasia (CAH) Autosomal recessive disorders that cause the impaired synthesis of cortisol and other hormones from cholesterol by the adrenal cortex. In 90% of cases it is due to insufficiency of the genes coding for 21-hydroxylase, which causes: - a decreased amount of Aldosterone and as a result wasting of salt. - a decreased amount of Cortisol and as a result adrenal insufficiency. Decreased amount of Cortisol causes the increase of elements such as ACTH which increases the amount of Pregnenolone, that induce the conversion in progesterone and then testosterone. So as a result insufficient synthesis of cortisol and mineralocorticoids is accompanied by excessive synthesis of testosterone. In the classical version of this condition there is a virilization and salt loss in severe cases. There is a Genotype–phenotype association in CAH caused by 21-hydroxylase deficiency. The non-classical phenotype is not distinguished by virilization or salt loss. In case of mutation in the P39L gene, 70% of cases are associated with nonclassical conditions and 30% are with classical conditions. Because the minimal residual activity, despite the patients, is the same variation present a minimal residual activity of the enzyme. 2 patients may therefore present exactly the same combination of variations but with a different phenotype with different severity of the condition – this is because there are some variations of modifier genes that modify the activity of this enzyme. 2 The diagnosis of CAH is not done by using molecular genetics analysis but with a biochemical approach. The graph shows the serum concentration of 17-hydroxyprogesterone in several patients. This molecule should be converted into 11-deoxycortisol but couldn't be converted because of the deficit or error of the enzyme responsible for that. It is possible to measure the concentration of the precursor molecule at basal levels and after stimulation with ACTH – the clusters of patients can be seen that represent the classical CAH, non-classical CAH and the carriers of this condition. The incidence is not very rare. 1:10.000/1:14.000 live births. Why is this gene so frequently affected? This gene is located in a region which is prone to rearrangement, an unstable region. It is located near an unstable region coding for receptor HLA. Considering the region of this gene there are two blocks of duplication with a high homology. In one region there is gene coding for 21-Hydroxylase and in the other duplicated segment there is a pseudogene, so a region that represents a very high homology, but it doesn't work. A pseudogene contains a very large number of mutations that disrupt the function and cause no functioning of the sequence. As a conclusion there are a gene and a pseudogene in the same region that represent a very high homology. Because of this high homology during meiosis it can occur a non-equal pairing of sister or non-sister chromatids. This results in different processes: Unequal Exchange of Chromatid (UEC) or Unequal Exchange of Sister Chromatid (UESCE) or Gene Conversion. 25% of mutation are due to UEC or UESCE – can cause either a fusion of the gene with the pseudogene (causes inactivation of the gene) or a deletion of the entire gene. 75% is due to this micro homology between pseudogene and gene which induces a mechanism called gene conversion – some sequences of pseudogene are donated to the normal genes. So after gene conversions normal genes contain mutations that normally are present in pseudogene, it doesn't work anymore. 3 One of several models that can explain gene conversion is the presence of this high homology. The formation of heteroduplex after the pairing is recognized by a mismatch repair machine, then the normal sequence is changed into pseudogene sequence. At the end there are some sequences of pseudogene which are now present in the normal gene. It is important to know the mechanism because it helps the molecular diagnosis of this condition. The best diagnostic strategies for such a disease are those to sequence the specific targeted regions to define the mutation. If a specific mutation is found, the parents should also be checked to see if the mutation is inherited or not. For example: in these two cases one presents the condition and the other does not. It is expected to find two variations in an affected individual. Because of the mechanism being gene conversion, more than one variation in cis can be acquired, therefore, to discriminate if the variations are in trans or in cis it is necessary to loos for the parents. In the second case the mother has both variations in cis because the daughter has both variations. In the first case the variations are trans one of each inherited from a different parent. As a disease is being studied, it is important to consider that there can be a unique condition in different patients, because the pathogenetic mechanisms can be very different in different regions. In Caucasian populations bimodular condition is present only in 69% of individuals. 17% of individuals have only the normal gene – no possible mispairing between pseudogene and the gene. There is also a small fraction of individuals that represent trimodular configuration, in which there is a block of pseudogene, a block of normal gene and a block of the gene with mutation. So by sequencing these genes individually, the patients appear as a carrier of mutation, but they are not because there is a normal copy of the gene. This is the reason why in this case report the researcher expected to find a phenotype in the fetus. Because the fetus carries two different variations, one maternal and one paternal. But the babies born do not present any problem. Considering the genome structure of the region they found a trimodule condition in which the father explains the lack of clinical condition in the daughter because there is a mutation in one block but there is also a normal block in cis – so this individual is a carrier but isn’t affected. Additional material: 4  6. Loss of function in dosage sensitive genes In the case of dosage sensitive genes a dominant scenario is expected – the family tree would show an affected individual every generation. In this case 50% of products do not avoid the phenotype – important increase of pathogenesis. As an example of the dosage sensitive gene there are Williams Beuren Syndrome (WBS) and Smith Magenis Syndrome. The difference between these two syndromes is that the deletion that causes the WBS is a contiguous gene, which means there are more than one dosage sensitive genes that together cause this condition. On the other hand in SMS there is only one dosage sensitive gene involved. WBS and SMS are due to deletion of dosage sensitive genes. An increased copy number of these deleted regions causes a phenotype that is carrying different clinical symptoms. For example in WBS the patients are very social, and conversely the duplication of this region causes autism causing problems with verbal communication. Most of the time the deletion and duplication represent two different phenotypes. Usually the phenotype due to the duplication is less severe than the phenotype due to the deletion. The frequency of patients with deletions is higher than the frequency of the patients with the duplication because the phenotype is milder and sometimes undiagnosed. WILLIAMS BEUREN SYNDROME - Genetics features 5-6% of all cases of ID of genetic origin - Incidence: 1/7,500-10,000 5  - Sporadic inheritance The patients represent very peculiar physical and clinical characteristics. Most of the time the physician can recognize the patients by looking at the face. Physical and clinical characteristics: - Facial dysmorphisms - Connective tissue anomalies - Congenital vascular and heart disease (Supravalvular aortic stenosis) - Hypertension - Coordination deficit - Hyperacusis Infantile - Hypercalcaemia - Impaired glucose tolerance - Sensorineural Hearing Loss Cognitive features: - IQ 40-80 - Visuospatial deficit - Language strength Behavioral features: - Loquacious - Sociable & Friendly - Attention deficit - Hyperactivity - Anxiety WBS Genomic Region Sporadic and familial Williams Syndrome result from deletions of genetic material from adjacent (contiguous) genes located on the long arm (q) of chromosome 7 (7q11.23). It is definite that this gene is involved in cardiac problems considering the clinical characteristics. The regions that are deleted involve several genes. In this case more genes are involved in pathogenesis of this condition. Also other genes seem to be implicated despite this being only confirmed via animal models such as mouse models (see the table below). Recently by using fibroblasts of patients and the formation of pluripotent stem cells after differentiation in the cortical neuron it is studied the expression of the genes present in cortical neurons. The result of the study is that the more genes involved in the WBS the dysregulation of several genes that are involved in neurodevelopment increase. Downregulation of other genes outside the region in WBS Under the recent works regarding the analysis of RNA in the peripheral blood of patients it was found that several genes are downregulated compared to the control gene. Interestingly the downregulation found not only in the region involved but also other regions involving other genes. The genes that are half regulated in other regions of the genome are due to the downregulation of microRNAs, which normally control the regulation of the genes. 6 Dysregulation of the mRNA/miRNA network involving genes outside of the 7q11.23 region is likely related to the complex phenotypes observed in WS patients. So this condition can be considered as down syndrome of the genomic region. SMITH MAGENIS SYNDROME Smith Magenis Syndrome (SMS) involves only one gene, RAI1, which is dosage sensitive and regulates other genes during the development, so also in this case multiple genes result involved. The incidence of this condition is now around 1:15.000. SMS is characterized by a peculiar craniofacial and skeletal dysmorphism associated with other clinical signs, clinicians recognize these patients due to their severe psychiatric manifestations such as aggressive behavior and autistic traits. These patients present an intellectual disability which can be moderate or severe and they are characterized by sleep disturbance. In this syndrome the sleep disturbance is represented by early waking and daytime napping, in most of the patients it is also found an inversion of the melatonin cycle, indeed, measuring the melatonin in control and SMS patients, it is found that the secretion of melatonin is increased during the day (it should be the contrary). Melatonin secretion is important to regulate the circadian cycle, it is controlled by a series of genes which are expressed in an oscillatory way starting from the hypothalamus region to the peripheral cells. SMS is a genomic disorder produced by a deletion mediated by the presence of segmental duplications causing both deletions and duplications in the same event. 90% of the patients present a deletion of about 4 Mb containing 28 genes but the single gene which is implicated is the red one (see photo). It is important to remember that the duplication of this gene causes the Potocki-Lupski Syndrome. 7 In only 10% of patients has been discovered a loss of function mutation in this gene and the affected patients present the same phenotype of the ones carrying the deletion. In the table you can compare the phenotypes of patients carrying the deletion and the ones carrying the mutation: cannot distinguish the two except some other features such as cardiovascular or renal tract abnormalities in patients with deletions. RAI1 belongs to the epigenetic machinery, it is defined as a “reader”. It has been found that RAI1 complex can read and identify the methylated histones and it is able to trigger the proteins implicated in the methylation of histones, thus activating the genes. In mouse models it has been demonstrated that the genes activated by this complex are related to neurodevelopment and maintenance and regulation of circadian rhythm, indeed they found that most of the genes involving the circadian rhythm are downregulated in mouse models. RAI1 is a dosage sensitive gene and for this reason its duplication causes another syndrome, the Potocki-Lupski Syndrome (PTLS), which involves several symptoms. When you consider patients with a suspected SMS only 50% reach molecular diagnosis, because the other 50% don’t present any deletion or mutation on RAI1. This can be explained with the findings of deletions in other genes or the presence of other syndromes which can downregulate RAI1. Recently, a loss of function mutation was found in a gene coding for a protein involved in the regulatory complex, this leads to a similar phenotype. For these reasons, we use array-CGH to detect the deletion, then if the deletion is not present, the genes presenting similar phenotypes, if mutated, are analyzed with NGS panel and you need to exclude microdeletions by MLPA analysis. If the tests are positive, you confirm the diagnosis while if negative, you can use RT-qPCR or high-resolution array CGH and WES to identify new genes involved. In the case report we have a proband with SMS phenotype. Normally it is difficult to reach a diagnosis in infancy and only during adolescence the symptoms are well defined (behavior and facial conformation). 8 Case report The proband is negative for the deletion but it is found a micro deletion on exon 5 which affects part of the exon 6 of RAI1 gene. The protein lacks the last domain which is important for its function. It is also found that the proband has an upregulation of the RAI1. Measuring the quantitative expression of the relatives and the proband it was observed that the overexpression occurs also in the mother and the brother. Evaluating only the wild type allele of the patient you can find that only half amount of the normal product is produced, thus this confirms the diagnosis of SMS. However, the patients present 1/3 of normal transcripts and 2/3 of abnormal transcripts and this is the reason why she has SMS and not PTLS. The family presented the overexpression of RAI1 which should be linked to PTLS but they are not affected, this can be explained by the presence in cis of a missense variation in exon 3 which is very rare, and it is considered a benign variation. The mutated region is a site of recognition for transcription factors, which can be activator or inhibitor. However, we don’t know exactly the effects of this mutation and it is still under study. The mother and the brother don’t show any sign of PTLS, this can be due to reduced penetrance or variability of expression. The mother has some similarities with patients of PTLS in craniofacial dysmorphism, but she is not recognized as PTLS patients when analyzed by a specific program which recognizes the diseases based on facial dysmorphism. 7. Gain of function - altered product Another effect that we can define as a gain of function is when the variation causes an alteration of the product which interacts with a normal product neutralizing some of the functions of it. This is an antimorph variation, causing dominant negative effects. If a product in a complex interacts with other normal proteins, all the complex doesn’t work. An example is the osteogenesis imperfecta which is primarily characterized by bone fragility, but we can observe a very wide range of phenotypes from a less severe bone fragility to incompatibility with life (type 2 OI). In 90% of the cases the genes which are involved in osteogenesis imperfecta are the ones involved in type 1 collagen fibrils (COL1A1 and COL1A2) which code for some chains building the triple helix of collagen fibrils. There are different steps when the synthesis occurs: the expression of the genes, 9 production of two 𝛼1 chains and one of 𝛼2 chain (thus you need different regulation for the two genes) and the assembly of the three procollagen chains. The chain is assembled outside where it undergoes further modification. The wide range of phenotypes can be explained through a severe mutation which inhibits the expression of a gene and the molecule is normal, this explains the milder phenotype. In a severe variation you have a mild phenotype. The severe condition is due to the presence of missense mutation which causes the formation of an abnormal chain which has to interact with the normal one and so the molecules are not properly folded to form the fibrils. If the mutation occurs near the carboxy terminal the effect is more severe than one occurring in the terminal part of the molecule, so the position of the mutation is important, too. Of relevance is also the amino acid that substitutes the normal one. The fibrils of collagens have a peculiar organization: in the first position we always have a glycine as the first amino acid, this is very important, a mutation there causes a severe pathology. Also in this case we can find that for the same mutation different phenotypes are shown. The severity is then influenced by which chain has the mutation, one of the two chains is requested in a double dose in relation to the other. Usually these mutations are dominant, but a small portion of patients have been found to possess a recessive form, this is explained with mutations in genes encoding the proteins involved in production or maturation of the collagen, thus indirectly involved in collagen formation. 10