MOLECULAR BASIS OF DISEASES.

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1 Molecular Basis of Diseases 1 MOLECULAR BASIS OF DISEASES. Assembled by: Prof. Janos Szabad University of Szeged, Department of Biology Szeged, April 2010 INTRODUCTION Life of every human starts with fusion of female and a male germ cell. There are normally 23 chromosomes in each and every of the haploid (n) germ cells. The zygotes are diploid (2n) with a maternally- and a paternally-derived set of chromosomes. Genetic constitution of any human being is established upon formation of the zygote. The altogether 46 chromosomes are classified into two groups. One includes the X and the Y, the so-called sex chromosomes. The others are the autosomes (Fig. 1). While every cell of the females carries two X chromosomes (XX), there is an X and a Y sex chromosome (XY) in cells of the males. There are two sets of autosomes in each cell. The autosomes (as well as the X chromosomes) can be arranged into pairs of the so-called homologous chromosomes. The homologous chromosomes are of the same size, organized in the same manner and carry genetic information for the same trait in their identical sites. The genetic information may be identical or different (Fig. 2). Figure 1. The human chromosome set. (Anaphase chromosomes.) There is a single DNA molecule in each of the chromosomes. The DNA in the 22 autosomes as well in the X and in the Y chromosomes comprises the human genome. The human genome consists of 3x10 9 base pairs that altogether would make up a 93.5 cm long DNA thread. Sequence of the nucleotides in the human genome has been known since There are about 30,000 genes in the human genome. Figure 2. The mode how we cross our arms is determined by alleles of a gene linked to one of the autosomes. Those who keep the left arm above the right arm are homozygous for the recessive allele. People with the right arm above the left arm are homo- or heterozygous for the dominant allele. Less than a century ago, diseases of largely nongenetic causation accounted for the great majority of death in children. As a consequence of the vast improvement in public health, genetic diseases have come to account for an increasing percentage of deaths among children. For example, pediatric deaths due to genetic reasons increased from 16.5 % in 1919 to 50% by 1976 and this proportion keeps on increasing. Thanks to the immense development of genetics and molecular biology, the human genome has been completed and we are aware of 18,000 autosomal, more than 1,000 X-, 57 Y-linked traits and 63 associated with the mitochondrial DNA. Knowledge concerning the DNA-related genetic diseases not only offers appropriate ways for treatments but also for prevention. The present overview aims to present examples, along a scheme, to describe the relationship between DNA, genes and life of the cells and genetic diseases. When do cells feel good? Cells feel good once they are diploid and at least one of the maternally- and the paternally derived copy of the genes is normal, functions well. Cells, in general, are unhappy if their genetic material is unbalanced, i.e. (i) some of the chromosomes or part of a chromosome is present in more than the usual two copies per cell or (ii) some of the chromosomes or part of a chromosome is missing. In fact, in most cases genetic imbalance results in death. It is a well-established fact that cells tolerate better the gain of chromosome or part of a chromosome than the loss of a chromosome or part of any chromosome. Once the number and organization of the chromosomes are perfect in the cells, it is the conditions of the genes that determine how well cells feel, that is the nature of the mutant alleles in the cells. There are plenty of so-called polygenic traits (diseases if you wish) that are determined by combinations of several alleles of several genes. Typical examples are heart diseases, some types of diabetes and so on. Environmental factors play major roles in the polygenic diseases. More and more are known concerning the roles of the environmental factors. The guideline: chromosome and gene mutations The origin of spontaneous changes in the DNA is an inherent feature of the material for inheritance. Rate of the spontaneous mutations is 10-9 /base pair/replication. Mutations, sudden and heritable changes in the DNA,

2 Molecular Basis of Diseases 2 are also caused by physical, chemical and biological factors. Mutations provide the basis for variability of the organism and of evolution. However, the mutations have unpleasant, frequently detrimental consequences. Mutations can be classified as summarized in Table 1. The guideline of the present chapter will follow the classification presented in Table 1. The second cases are consequences of mitotic nondisjunction and/or chromosome loss that take place in diploid (2n) cells in course of mitoses. Mitotic nondisjunction and/or chromosome loss lead to the formation of those types of the so-called genetic mosaics in which cells of an organism carry different numbers of chromosomes. (See the power point file of the lecture.) Table 1. Classification of mutations Chromosome mutations Changes in chromosome number (due to nondisjunction and/or chromosome loss) Structural changes (due to mostly chromosome breakages) Euploidy Aneuploidy Translocation Transposition Inversion Duplication Deficiency Autosome trisomy Of all the autosome trisomies, trisomy 21 is far the most frequent: it happens in 1/800-1/1,000 newborns. People with trisomy-21 posses symptoms of Downsyndrome. Some are summarized in Fig. 3. The source of Down syndrome is usually a nondisjunction in the mother in the course of egg cell formation and thus the formation of egg cells with two of the 21 st chromosomes. Point- or gene mutations Frame shift mutations Base pair substitution Addition Deletion Transition Transversion Changes in the number of chromosomes Euploidy The euploid cells carry multiple copies of the basic chromosome number (n). While the tri- (3n), the tetra- (4n) and the hexaploid (6n) plants have a profound importance in crop production, the triploid human newborns are very seldom (1/10,000 births) and are short-lived. They arise usually through the fertilization of single egg cells by two sperm. Triploidy amounts to about 15% of the chromosome number abnormalities that come about during fertilization. Most of the triploid embryos are aborted during the first two trimesters of pregnancies. Tetraploidy is a lot less frequent than triploidy. There were only very few cases reported and the newborn tetraploids lived very short. Aneuploidy In aneuploidy, the chromosome number of the cells departs by usually one from the normal 2n. They are usually trisomy (2n+1) and monosomy (2n-1). Monosomy is a lot less frequent than trisomy. Aneuploidy may include (1) all or (2) only some of the cells in an organism. The first cases emerge when a germ cells with normal haploid chromosome number (n) fuses during fertilization with a germ cell with one more (n+1) or one less (n-1) chromosome than normal. The aneuploid germ cells are consequences of (i) nondisjunction in the course of the first or the second meiotic division and/or (ii) chromosome loss during meiosis. (See the power point file of the lecture.) Figure 3. Some characteristics of Down-syndrome. All three chromosomes were isolated from a number of Down-syndrome people and their DNA was sequenced. Analysis of the DNA nucleotide sequences clearly revealed that every gene in all the three 21 st chromosomes were normal and thus not mutation(s) in the nucleotide sequence but altered gene-dose relationships are responsible for the formation of the abnormalities. There is 50% more gene product than normal in some of the 21 st chromosome-linked genes in cells of the Down-syndrome people. The excess gene products alter organization and/or function of the cells and thus lead to the formation of Downsyndrome. Several of the genes have been identified and their function described in the altered cell functions. Extra copy of the DYRK1A gene (that encodes for a kinase) is responsible for mental retardation, and loss of memory. The extra copy of the APP (amyloid precursor protein) gene is the reason for development of the Alzheimer disease by the age of 40 in practically all the Down-syndrome people. The phenomenon that an extra copy of a normal gene results in defective cell function is known as triploabnormality.

3 Molecular Basis of Diseases 3 So-called Down mosaics develop if gain of the 21 st chromosome happens through mitotic nondisjunction (Fig. 4). Features of the Down-syndrome traits appear in mosaic spots in some of those people. It should be mentioned that trisomy-21 is a predisposition to acute leukemia. Figure 4. Palms of a Down mosaic. Note the single and straight simian line ( ) on the palm on the hand side. The simian line pattern is normal on the palm on the right side. The risk of Down-syndrome increases exponentially with the maternal age (Fig. 5). Genes included in the heterochromatinized regions are inactivated. However, the genes below the telomere of the short chromosome arm, in the so-called pseudoautosomal region remain active. The pseudo-autosomal region is about 2.5 Mbp in size and includes a small number of genes. Although there is a single X chromosomes active in the XXX and in the XXXX cells (with two and tree Barr bodies, respectively), genes in the pseudo-autosomal region remain active and thus instead of the usual two these genes there are present in three and four copies in the XXX and in the XXXX cells. People with the XXY sex chromosome composition possess symptoms of the Klinefelter-syndrome (Fig. 6). There is roughly one Klinefelter-syndrome among 500-1,000 newborns. Their mental and learning abilities are slightly reduced. The extra X chromosome in their cells is of maternal origin in roughly 50% of the cases. As the nucleotide sequence of X chromosome pseudoautosomal region is very similar to that of the corresponding region of the Y chromosome, the triploabnormal condition for some of the genes in the aforementioned region well may account for abnormalities seen in the Klinefelter-syndrome people. There have been reports on XXXY and XXXXY Klinefelter-syndrome people reported. About 15% of the Klinefelter-syndrome people are Klinefelter mosaics. Some are XY/XXY others are XY/X0 types of mosaics. Figure 5. Relationship between maternal age and the risk of Down-syndrome children in live births. Edwards-syndrome stems for trisomy for the 18 th, and Patau-syndrome for of the 13 th chromosome. (It worth mentioning that about 20% of the Patau-syndrome people are Patau mosaics.) Sex chromosome trisomy Trisomy of the X chromosome (XXX instead of the usual XX) appears with a frequency of about 1/1,000 among the females. They are slightly taller than normal, their menstrual cycles are usually abnormal, possess reduced fertility and mental capacities. About 90% of the XXX females originate as the consequence of nondisjunction in the mother. The frequencies of the XXX newborns increase with maternal age, as described for Down-syndrome. Occasionally girls are also born with XXXX, XXXXX and even more X chromosomes/cell. In general, the more X chromosomes they carry, the weaker are their physical and mental abilities. The basis of the XXX abnormalities lie - once again - in altered gene dosage conditions. In the XX cells, one of the X chromosomes is almost completely inactivated through heterochromatinization, Barr body formation. Figure 6. Characteristics of the Klinefelter-syndrome men. There are XYY men also known. They appear at a frequency of about 1/1,000 men and emerge mostly due to nondisjunction in XY men during spermatogenesis when sperm with two Y chromosomes form. Sperm with YY chromosomes can also form in XYY men

4 Molecular Basis of Diseases 4 through secondary nondisjunction during spermatogenesis. [During secondary nondisjunction, aneuploid germ cells (e.g. YY) derive from the already aneuploid (e.g. XYY) germ-line cells.] The XYY men are usually taller than normal and their mental abilities are slightly reduced. They are characterized by hyperactivity, reduced abilities to concentrate and study. Autosome monosomy Monosomy of the autosomes (the 2n-1 condition), is incompatible with human life once it would cover every cell in the body. However, groups of monosomic cells may occasionally survive. For example, cells monosomic for the 22 nd chromosome may survive in the nervous system and can be the sources of meningiomas. Sex chromosome monosomy Monosomy for the X chromosome has been known as Turner-syndrome and occurs at a rate of 1/2,000-1/3,000 among the newborn girls. (The X0 zygotes amount to about 1-2% of the conceptions, i.e. over 99% of the X0 zygotes are aborted.) Features of the Turner-syndrome people are summarized in Figure 7. Their origin is related to the lack of sex chromosome in the sperm in about 60-80% of the cases. About 30-40% of the Turner-syndrome females are Turner mosaics, mostly XX/X0, however some are XY/X0. change when parts of chromosomes (with many-many genes inside) or even a single gene copy will be present in more than two, or less than two copies. Examples for gain or loss of parts of chromosomes will be discussed now and then the single gene cases will be covered. The guideline is summarized in Table 1. Translocations and genetic imbalance Translocation is the interchange of genetic material between non-homologous chromosomes. There are different types of the translocations. In the example illustrated on Figure 8, part of a 5 th chromosome became translocated onto a 13 th chromosome. The amount of heritable material is normal in cells carrying both the shortened 5 th and the longer than normal 13 th chromosomes. (The translocation is in a balanced condition.) However, complications emerge when germ cells of such a human fuse during fertilization with germ cells with a normal set of chromosomes (Fig. 8). Genetic imbalance commences in two of the combinations. Part of the 5 th chromosome is missing in one of the combinations, a phenomenon called in general partial monosomy. New born children in which part of the 5 th chromosomes is missing possess symptoms of the so-called Cri-du-chat syndrome and are sort-lived. Children in whom part of the 5 th chromosome is present in three copies (partial trisomy) live only few years (Fig. 8). condition Balanced translocation Germ cells Figure 7. Features of Turner-syndrome. The reason for Turner-syndrome is once again the altered gene dosage relationships in the cells: there is a single copy of the X chromosome pseudo-autosomal region in the X0 cells instead of the usual two. The short statue is related to the lack of one copy of the SHOX gene that encodes the formation of a transcription factor. This transcription factor controls growth of the limb buds in the embryos. The lack of one SHOX gene copy and the associated defect is a typical example of haploinsufficiency. For normal function of the limb bud cells normal function of both the maternally and the paternally-derived SHOX gene copies are required. The examples introduced above are all related to altered gene dosages due to changes in chromosome number. Naturally, the gene dosage relations can also Type of progeny Balanced translocation Loss of part of the 5 th chromosome Cri-du-chat syndrome Duplication of part of the 5 th chromosome Dies in childhood Figure 8. A 5 13 translocation and its consequences. The grey-colored chromosomes derive from germ cells with normal sets of normal chromosomes and fuse during fertilization with germ cells of the translocation-carrying human.

5 Molecular Basis of Diseases 5 In reciprocal translocations two different chromosomes break and their parts are mutually replaced (Figs. 9 and 10). (On average one in 625 newborns carry a reciprocal translocation.) In lucky cases, when the breakages take place in gene-free regions, the reciprocal translocations have little if any consequences. However, it happens occasionally that the breakages destroy (or alter) function of one of both of the affected genes and lead to severe consequences (Fig. 9). In such cases the translocations identify important genes and allowed their positional cloning to acquire molecular function the normal gene. the another chromsome is present in three copies (partial trisomy; Fig. 10). Fate of the zygote depends on the genes that are present only in one and also on those present in three copies. The simultaneously partially monosomic and partially trisomic combinations are lethal in the vast majority of the cases. In case of the so-called Robertsonian translocations the short arms of two non-homologous chromosomes are lost and the two long arms fuse at their centromeres to form a single Robertsonian translocation (Fig. 11). Figure 9. The illustrated type of reciprocal translocation between chromosomes 9 and 22 frequently results in myelogen leukemia, showing that one of the breaks altered function of a gene with essential function if life of some types of the white blood cells condition Reciprocal translocation Figure 11. The mechanism of the Robertsonian translocations. While long arms of two non-homologous acrocentric chromosomes fuse and form a Robertsonian translocation, the short arms are lost. Germ cells Type of progeny Normál Partially aneuploid germ cell Partially aneuploid germ cell Partial trisomy and partial monosomy Balanced translocation Balanced condtition Figure 10. Four different types of germ cells derive from germ-line cells that carry a reciprocal translocation. Fate of the progeny depends on the possible combinations of the unusual chromosomes. Four types of germ cells derive during meiosis with a reciprocal translocation (Figure 10). While one carries a normal chromsome, the translocation is in balanced condition in another one, two are partial aneuploids. Should these germ cells fuse with germ cells with a normal sets of chromsomes, in two of the four possible combinations part of one of the chromosomes is missing (partial monosomy) part of The Robertsonian translocations may include any two of the acrocentric chromosomes 13, 14, 15, 21 and 22. The short arms carry practically only the rrnaencoding so-called ribosomal genes in tandem arrangements. Although two sets of the ribosomal genes are lost during formation of a Robertsonian translocation, the remaining eight are plenty to ensure normal cell functions. However, the Robertsonian translocations lead to complications in the course of meiosis just as the other types of translocations: they hinder pairing of the homologous chromosomes during the first meiotic prophase and also their segregation. The Robertsonian translocations in which the 21 st chromosome is included leads frequently to the formation of germ cells with basically two of the 21 st chromosomes and thus such people have a high chance of Downsyndrome child born Transpositions imply transposition of chromosome segments onto another position in the same chromosome. Transpositions have little importance in human genetics. Inversion An inversion is the result of two breaks in the same chromosome and reinsertion of the fragment in an inverted order (Figs. 12 and 13). About one in a thousand human carries an inversion. Inversions, unless the break points disrupt gene functions, do not reduce viability of the cells. However, since the inversions interfere with pairing of the chromosomes

6 Molecular Basis of Diseases 6 during meiosis, lead to the formation of unusual chromosomes or chromosome fragments (Fig. 12. and 13). Pairing of a normal and a chromosome with a pericentric inversion (in which the inverted part includes the centromere), chromosomes form in which while some part is missing other is duplicated (Fig. 12). Should these chromosomes participate in fertilization, viability of the zygote is determined which genes are in partial monosomic and which are in partial trisomic conditions. Such zygotes are almost invariably lethal. In case of the paracentric inversions (in which the centromere is not included in the inversion), two unique products from as a result of a single crossing over during meiosis: (i) and acentric fragment that is lost as it does not contain centromere and (ii) a socalled dicentric fragment with two centromeres (Fig. 13). Not only there are genes missing from the dicentric chromosomes but, if they happen to get into a zygote, enter a so-called fusion-bridge-fusion cycle in the embryo and form chromatin bridges between the daughter nuclei. The chromatin bridges break and bring about further disturbances... Figure 12. Meiotic products resulting from a single crossing over within a heterozygous pericentric inversion loop. Duplication Duplication is the duplication of any chromosome region that contains one or several genes. Duplications originate usually from unequal crossing-over that occurs during meiosis between misaligned homologous chromosomes. The presence of three normal copies of some genes leads to abnormal cell functions, a phenomenon called triplo-abnormality. A good example is the Charcot-Marie-Tooth disease that is characterized by late childhood or early adulthood onset of progressive dystrophy of the distal limb muscles and touch sensation, predominantly in the feet and legs but also in the hands and arms in the advanced stages of the disease (Fig. 14). It affects approximately 1 in 2,500 persons and exists in several forms. The most common form is associated with a 1.5 Mbp duplication of the 17 th chromosome. It appears that three copies of the PMP22 gene is the reason for the Charcot-Marie-Tooth disease. The PMP22 gene encodes a component of the peripheral myelin. The increased dose of the PMP22 gene leads to demyelination and to development of the disease. Figure 14. Charcot-Marie-Tooth disease. Interestingly, loss of the PMP22 gene function (due to small deficiencies or loss-of-function mutations) leads to hereditary neuropathy with liability to paralysis. Increase by 50% or loss by 50% of the PMP22 gene is a typical example of the dosagedependent gene sensitivity known as or triploabnormal and haplo-insufficient gene functions. Figure 13. Pairing during meiosis of a normal and a chromosome with paracentric inversion. A crossing over leads to the formation of a dicentric chromosome and an acentric fragment. Deficiencies Deficiencies mean the loss of major DNA segments along with several genes from the chromosomes. (Deletions imply the loss of very few, frequently only one, base pairs.) Characteristic features develop once haplo-insufficient genes are lost in the deficiencies.

7 Molecular Basis of Diseases 7 The Turner- and the Cri-du-chat syndromes are typical examples of the deficiency-related diseases. A few more will be listed below. People showing symptoms of the Wolf-Hirschorn syndrome (Fig. 15) carry a deficiency that removed the distal short arm of chromosome 4 (4p16.3). The genes responsible for the abnormalities are WHSC1 and WHSC2. They appear to be engaged in regulation of cell cycle progression, cell adhesion and in regulation of RNA polymerase activity. Figure 15. Wolf-Hirschorn syndrome. The most common abnormalities include severe to profound mental retardation, seizures, poor muscle tone, cleft lip and/or cleft palate. Characteristic facial features include "fishlike" mouth, small chin, ear tags or pits, and cranial asymmetry. Characteristic features of the William syndrome originate due to loss of the 7q11.23 segment of the long arm of the 7 th chromosome (Fig. 16). Figure 16. The most common symptoms of Williams syndrome are mental retardation, heart defects and unusual facial features, low muscle tone high verbal and overly sociable abilities but also the lack common sense and inhibited intelligence. Patients tend to have widely spaced teeth, experience heart murmurs and the narrowing of major blood vessels as well as supravalvular aortic stenosis. There are 26 genes in the 7q11.23 region. Haploinsufficient conditions of a number of those are responsible for symptoms of the Williams syndrome. Researchers believe that the loss of several of these genes contributes to the characteristic features of this disorder. The ELN gene, which codes for the protein elastin, is associated with the connective-tissue abnormalities and cardiovascular disease (specifically supravalvular aortic stenosis and supravalvular pulmonary stenosis). Product of the CLIP2 gene associates with the growing ends of the microtubules. Reduced dose of the CLIP2 gene may contribute to the unique behavioral characteristics of the Williams syndrome people, learning disabilities, and other cognitive difficulties. Product of the GTF2I gene appears to be engaged in transcription regulation. Product of the GTF2IRD1 gene is a transcription factor with thus far unknown function. The LIMK1 gene seems to be engaged in cell proliferation control. Its haplo-insufficient condition is responsible for the cardiovascular abnormalities. In Prader-Willi syndrome an about 4Mbp of the long arm of chromosome 15 (15q11-13) is deleted. When such a chromosome is inherited from the father the child manifests a disease known as Prader-Willi syndrome (Fig. 17). Figure 17. Eight year old Eugenia Martinez Vellejo was paraded around on fairs as la monstrua because of her obesity. She was most likely affected by the Prader-Willi syndrome. (Painting by Juan Carreno de Miranda, 1680.) Prader-Willi syndrome is a very rare genetic disorder. Its incidence is between 1/12,000-1/25,000 live births. It is characterized by hypotonia, short stature, obesity, small hands and feet, almond-shaped eyes hypogonadism, infertility and mild mental retardation. Prader-Willi syndrome develops when the chromosome with the deficiency is of paternal origin and although the maternally derived 15 th chromosome carries the 15q11-13 region, it is inactivated, silenced through genomic imprinting. (It worth mentioning in brackets that genomic imprinting is a genetic phenomenon by which certain genes are expressed in a parent-origin-specific manner. It is an inheritance process independent of the classical Mendelian inheritance. Imprinted genes are either expressed only from the allele inherited from the mother or in other instances from the allele inherited from the father. Genomic imprinting is an epigenetic process that involves methylation and histone modifications in order to achieve monoallelic gene expression without altering the genetic sequence. There are about 200 loci in the human genome that are imprinted. The epigenetic modifications are established in the germ-line and are maintained throughout all somatic cells of an organism.) In the reverse case, where the deficiency carrying chromosome is maternal and the normal chromosome is paternal origin, Angelman syndrome develops (Fig. 18). Angelman syndrome people are characterized by developmental delay, speech impairment, balance disorder, tremulous movement of limbs and behavioral uniqueness such as any combination of frequent laughter/smiling, apparent happy demeanor; easily excitable personality, often with hand flapping movements, hypermotoric behavior, short attention. Figure 18. Angelman (also known as happy puppet) syndrome. Boy with a Puppet. Painting by Giovanni Francesco Caroto ( ); Castelvecchio Museum, Verona. The boy was most likely affected by Angelman syndrome. There a number of genes involved in development of the Prader-Willi syndrome: the SNRPN and the necdin genes along with clusters of snornas:

8 Molecular Basis of Diseases 8 SNORD64, SNORD107, SNORD108 and two copies of SNORD109, 29 copies of SNORD116 and 48 copies of SNORD115. The SNRPN gene encodes the formation of the small nuclear ribonucleoproteinassociated protein N that is one polypeptide of a small nuclear ribonucleoprotein complex and belongs to the snrnp SMB/SMN family. The protein plays a role in pre-mrna processing and possibly in tissue-specific alternative splicing events. Studies in mouse suggest that the necdin protein may suppress growth in the postmitotic neurons. The snornas are a class of small nucleolar RNA molecules that guide chemical modifications of other RNAs, mainly ribosomal RNAs, transfer RNAs and small nuclear RNAs. They are encoded by the SNORD genes. Studies of human and mouse model systems have shown that deletion of the 29 tandem-arranged copies of the SNORD116 snorna encoding genes is the primary cause of Prader-Willi syndrome. SNORD116 is a non-coding RNA molecule which is engaged in modification of other small nuclear RNAs (snrnas). This type of modifying RNA is usually located in the nucleolus, the major site of snrna biogenesis. SNORD116 belongs to the C/D box class of snornas that function in directing site-specific 2'- O-methylation of substrate RNAs. SNORD116 is expressed prevalently in the brain. Mouse models of Prader-Willi syndrome show similar symptoms to humans, i.e. hyperphagia and growth deficiency. Prader-Willi syndrome patients have high ghrelin levels, which are thought to directly contribute to the increased appetite, and obesity seen in this syndrome. Ghrelin, that that stimulates hunger, is a hormone produced mainly in the lining the fundus of the stomach, epsilon cells of the pancreas and in the hypothalamic where it stimulates the secretion of growth hormone from the anterior pituitary gland. Ghrelin levels increase before meals and decrease after meals. It is considered the counterpart of the hormone leptin, which induces satiation when present at higher levels. Abnormal ghrelin levels well may explain short stature and obesity. Types of the genes Abnormalities associated with complete or partial trisomy, with duplications, as well as with monosomy and deficiencies clearly showed that the genes can be classified into two major classes according to dose sensitivity: (1) some of the genes are dose-sensitive, others are (2) dose insensitive. (1) In the dose sensitive genes the cell functions depend on the number of gene copies and cells possess abnormal functions when the gene dose departs from the usual two (+/+) per cell. There are two types of the dose-sensitive genes: the triploabnormals and the haplo-insufficient ones. (Some of the dose-sensitive genes possess triplo-abnormalities once they are present in three copies per cell and haplo-insufficiency once there is only one gene copy in the cell.) As expression of the dose-sensitive genes does not seem to be regulated, concentration of the encoded gene products is proportional to the number of gene copies. When the genes are present in more (+/+/+) or less (+/ ) than the usual two (+/+) copies, the gene product relationships are altered leading to abnormal cell functions and serious consequences. (2) Most of the genes are dose insensitive. In these cases the gene function does not depend on the number of gene copies, doses and it is irrelevant to the cells whether they carry two, three or one copy of the gene. The reason for dose insensitivity is that expression of these types of the genes is regulated on several levels, most importantly on the level of transcription. Point- or gene mutations Point mutations are minor and heritable changes in the DNA. Once they happen inside the genes they are called gene mutations. There are two major types of point mutations: (i) the frame shift mutations originate through the addition (insertion) of usually one or the deletion by usually one base pair (Table 1). (ii) In the base-pair substitution mutations one base pair is replaced in the DNA by another base pair (Table 1). Although the gene mutations occur in single genes, they do not always disturb gene function. Most of the gene mutations, although not all, belong to the socalled loss-of-function class and leads to partial or complete loss of gene function. While the partial lossof-function types of mutations are rather frequent, the complete loss-of-function mutations (the so-called null alleles) are quite rare. The loss-of-function mutations are almost always recessive. Some of the gene mutations are of gain-of-function type. They are dominant, encode the formation of mutant gene products and can be classified into two major classes. The so-called dominant negatives (also called antimorphs) encode the formation of mutant gene products that eliminate function of the normal gene product. The so-called neomorph types of the gain-of-function mutations encode gene products that interfere with processes in which the normal gene product plays no role. The neomorph mutations are rather seldom. The dominant mutations can be classified as summarized in Table 2. Table 2. Classification of the dominant mutations (M) Type of the dominant phenotype mutation (M) M/+ M/+/+ Haploinsufficient Gain-offunction loss-offunction Dominant negative Neomorph Less severe than M/+ As severe as M/+ The + symbol stands for the normal, so-called wild type allele Type of the dominant mutation can be deduced on the basis of the M/+/+ condition, which is how the mutant phenotype is affected by addition of an extra, normal gene copy (Table 2).

9 Molecular Basis of Diseases 9 As mentioned above, there are genes that once present in three copies (+/+/+ instead the usual +/+) bring about triplo-abnormality. It can and does happen that mutations originate in the regulatory sequences of the genes and the mutation (i) leads to overexpression of the gene. Such conditions are reminiscent of the triplo-abnormalities. In some cases the mutations in the regulatory sequences (ii) bring about gene expression in cell types where the gene is normally not expressed. Such mutations well may have detrimental consequences. We ll turn attention now to consequences of the gene mutations. Point or gene mutations Point mutations represent minor, heritable changes in the DNA. Once happen within the frames of the genes they are called gene mutations. Most of the point mutations originate through (i) addition or deletion of usually single base pairs leading to the formation of frame shift mutations or (ii) by base pair substitutions (Table 1). The gene mutations usually disturb function of the genes, although this is not always the case. The vast majority of the gene mutations - however not all - are of loss-of-function type and lead to partial or complete loss of gene function. While the partial loss-offunction mutant alleles are rather common, the complete loss-of-function (also called null) mutant alleles are rather seldom. The loss-of-function alleles are usually recessive, but not always. Mutations may lead to the formation of gain-offunction mutant alleles that are of two types. The socalled dominant negative (also called antimorph) mutations encode the formation of mutant gene products that block function of the gene products encoded by the normal alleles. Products of the socalled neomorph mutations interfere with a different biochemical pathway in which the normal counterpart participates. The gain-of-function mutant alleles are almost always dominant. The dominant mutations can be classified as summarized in Table 2. Table 2. Classification of the dominant mutations Type of the dominant phenotype mutation (M) M/+ M/+/+ Haploinsufficient gain-offunction loss-offunction Dominant negative Neomorph Less severe than M/+ As severe as M/+ Note: the + symbol stands for thee normal, wild-type allele. Nature of the dominant mutation can be established through phenotype of the M/+/+ condition, i.e. in presence of an additional normal gene copy (+; Table 2). As mentioned earlier, extra copies of normal genes (e.g. +/+/+ instead of the usual +/+) lead to the formation of triplo-abnormality. Similarly, mutations in the regulatory sequences may lead to overexpression of the genes (the so-called hypermorph mutations) or expression in unusual cell types. Such events can also disturb cell functions with severe consequences. The following sections of the chapter will show examples for point mutation-related abnormalities. Autosomal dominant mutation Polydactyly is a congenital physical anomaly in humans having supernumerary fingers or toes (Fig. 19). Its incidence is two in every 1,000 live births. Neither penetrance nor expressivity of the mutation is 100%. Mutations in a variety of genes can give rise to polydactyly. Mutations in the LMBR1 gene are best characterized. The LMBR1 gene (Limb region 1) codes the formation of a transmembrane protein engaged in manifesting action of the sonic hedgehog morphogen involved in digit formation. Figure 19. Polydactyly. Another well known example for autosomal dominant mutations is the one that causes Marfan syndrome, a genetic disorder of the connective tissue. People with Marfan syndrome are typically tall, with long limbs and long thin fingers. The most serious complications are the defects of the heart valves and aorta, the dural sac surrounding the spinal cord, skeleton and the hard palate (Fig. 20). The mutation may also affect the lungs, eyes and vision. Nearsightedness and astigmatism are common; dislocation of the crystalline lens in one or both eyes is quite typical (Fig. 20). The best described cases of Marfan syndrome are caused by dominant mutations in the FBN1 gene which encodes the connective tissue protein a structural support for tissues outside the cell called fibrillin-1. The mutant gene products, encoded by dominant negative alleles, disturb function of the normal ones and thus typical symptoms develop. Fibrillin-1 also binds to transforming growth factor (TGF- ), that controls proliferation, cell differentiation and other functions in most cells. TGFacts as an antiproliferative factor in epithelial cells and at early stages of tumor formation. TGF- has deleterious effects on vascular smooth muscle development and the integrity of the extracellular matrix. It is generally accepted that the presence of abnormal fibrillin-1 leads to excessive TGF- in the lungs, heart valves and aorta weakening these tissues and causing Marfan syndrome.

10 Molecular Basis of Diseases 10 Figure 20. Features of Marfan syndrome. The lecture covered the above material Yet to elaborate - Somatic mosaicism and the ocogenes - Example for X-linked dominant mutations - Example for X-linked recessive mutations - Example for Y-linked mutations - Closing remarks