Theodor Boveri and Walter Sutton individually carried out research in the field of genetics and came up with the 'chromosome theory of inheritance' in the early twentieth century which was a follow up of Gregor Mendel's work. The theory stated that genes are found at specific loci on chromosomes, which are the source of genetic inheritance.
In diploid organisms, somatic cells(not germiline cells) possess two copies of the genome. Each autosomal gene(not sex gene) is therefore represented by two copies, or alleles, with one copy inherited from each parent at fertilisation. For the vast majority of autosomal genes, expression occurs from both alleles simultaneously. In mammals however, a small proportion (<1%) of genes are imprinted, meaning that gene expression occurs from only one allele[, process known as genomic imprinting.
What is genomic imprinting?
Genomic imprinting is an example of Non-Mendelian inheritance. It represents a type of epigenetic variation involving heritable changes which affect gene expression. It is a biological process whereby genetic alleles are labelled as descendant from either the father or the mother (parent-of origin specific imprints). Some imprinted genes are expressed from the maternally inherited chromosomes and others from the paternally inherited chromosomes. More than just 'marking' alleles, these imprints have a functional consequence resulting in monoallelic expression. It should be mentioned however that expression is not 100% monoallelic and in actual fact, expression is said to be favoured from one parental locus over the other, leading to unbalanced biallelic expression. This is a fundamental systematic procedure for normal mammalian development.
How imprints are manifested?
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DNA methylation is the main way of imprinting genes although other ways of epigenetic alterations such as histone modifications and nucleosome positioning (Henry Chung et al.) are viable. CpG dinucleotides, which lie upstream of the genes and which are clustered in regions called CpG islands(, are the victims of methylation. More than 50% of gene promoters in ubiquitous cells are linked to CpG islands and are normally unmethylated. Tissue-specific factors initiate the methylation process and, consequently, genes are silenced by blocking the binding of transcription factors to promoters, recruiting methyl CpG-binding proteins that compete with transcription factors or altering nucleosome formation, thus hindering transcription. One of the mechanisms of cytosine methylation is the recruitment of methyl-CpG-binding domain proteins (MBD). During the process of gene imprinting, there is sex-specific methylation and therefore serves as a way to differentiate between maternal and paternal genomes (Li E et al. 1993). Because of the fact that only one allele is expressed, imprinted genes are vulnerable to mutations lead to disastrous results. The establishment of imprints is a complex procedure that involves reprogramming of the entire genome.Reprogramming is of primary importance because accurate imprints must be passed on to the next generation. In other words, in males, all cells contain one set of chromosomes with male imprints (from the father) and another set with female imprints (from the mother), but when these chromosomes are passed on to the next generation, both sets in the germ cells must be reprogrammed to contain male imprints which account for paternal contribution. Likewise, in females, all cells contain one paternal set of chromosomes with male-specific imprints and a maternal set with female specific imprints, which are later reprogrammed in the germ cells to contain female-specific imprints exclusively so that they can be passed on to the next generation with maternal methylation patterns. On some occasions there is loss of imprinting which can result in the manifestation of several types of diseases or disorders. In this review we are going to look at the Prader-Willi Syndrome(PWS).
What is Prader Willi Syndrome?
Prader Willi syndrome is a genetic disorder, afffecting one in 20000 births and was first discovered in 1956 by Swiss doctors, Prof. A Prader, Dr A Labhart and Dr H Willi. They defined the disease based on specific clinical features:
Hypotonia: weak muscle tone, and floppiness at birth.
Hypogonadism: immature development of sexual organs and other sexual characteristics.
Obesity: caused by excessive appetite and overeating (hyperphagia), and a decreased calorific requirement owing to low energy expenditure levels. (Obesity is not normally a feature of those whose food intake is strictly controlled.)
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Central nervous system and endocrine gland dysfunction: causing varying degrees of learning disability, short stature, hyperphagia, somnolence, and poor emotional and social development.
Many people with PWS also exhibit characteristic facial and other physical
features. These include: almond-shaped eyes, a narrow forehead (measured across), a down-turned mouth with a triangular-shaped upper lip, and small hands and feet.
PWS represents a significant fraction of the neurodevelopmental cases linked to genetics and is one of the main causes of obesity. It is also responsible for serious physical, psychological and social handicaps.
Causes of Prader-Willi Syndrome
Interstitial 5-7 Mb deletion within the paternally inherited chromosomal 15q11-q13 region, an occurrence observed in 70% of cases.
Maternal uniparental disomy 15, situation resulting from a non-dysjunction of maternal chromosomes at meiosis and the child receives both of the chromosomes from the same parent. It can be either heterodisomic UPD (meiosis I) or isodisomic UPD (meiosis II or postzygotic chromosomal duplication), accounting for about 25% of cases.
Imprinting defects in the 15q11-q13 region observed in 3-5% of cases.
Mutations in the 15q11-q13 region, less than 1% of cases.
In addition to clinical features, diagnosis can be confirmed by genetic analysis of DNA methylation. It happens to be the only method able to detect all three of the above causes of PWS and also used to distinguish between Angelman Syndrome (which involves the deletion of the UBE3A gene on the maternal chromosome 15) and Prader-Willi Syndrome (Driscoll DJ, Waters MF, Williams CA et al. 1992)
Most deletions responsible for PWS can be detected by cytogenetics. The genes responsible are grouped over a distance of 2 million base pairs, which represent half of the 15q11-q13 region. However about 5% of cases of PWS are caused by micro deletions in the imprinting centre(IC) and cannot be detected. Further molecular analyisis of these deletions has led to the categorisation of three principal break sites in the 15q11-q13 region; BP1 (breakpoint 1) found nearer to the centromere, BP2 and BP3, which is nearer to the telomere(Amos-Landgraf JM, Ji Y, Gottlieb Wet al. 1999).
The structure of the 15q11-q13 region
The 15q11.2-q13 region can be roughly divided into four distinct regions (Figure 5): (1) a proximal non-imprinted region between BP1 and BP2 containing four biparentally expressed genes; (2) a 'PWS paternal-only expressed region' containing five protein coding genes (MKRN3, MAGEL2, NECDIN, and the bicistronic SNURF-SNRPN), a cluster of five repetitious snoRNA genes (HBII-436, HBII-13, HBII-438, HBII-85 and HBII-52) and several antisense transcripts (including the antisense transcript to UBE3A); (3) an 'AS region' containing the preferentially maternally expressed genes UBE3A and ATP10A and (4) a distal non-imprinted region containing a cluster of three GABA receptor genes, the gene for oculocutaneous albinism type 2 (OCA2) and the HERC2 gene. The exact function of each of these genes in the PWS phenotype is still being elucidated. The various created gene knock out mouse models will be useful in uncovering these contributions. (Mihaela Stefan et al. 2005)
Here we will be looking at the 'PWS paternal-only expressed region' in particular and how the different genes may potentially contribute to the Prader-Willi phenotypes.
Genes related to PWS region
Interestingly, singlegene mutations have not been found to cause PWS, suggesting that loss of multiple genes is required to produce this syndrome. However, it is likely that loss of expression of specific, individual genes may contribute to the various distinct phenotypes associated with this complex disorder. For this reason, it has been important to study the genes within the Prader-Willi interval individually.
C15orf2 is an intronless gene found between the Necdin (NDN) and SNURF-SNRPN genes in the PWS region. In 2000, C15orf2, which encoded an 1156-amino acid protein, was identified by Farber C et al. They used Northern Blotting to show expression of this particular gene in testis only. However this was reinvestigated to show, by reverse-transcription-polymerase chain reaction (RT-PCR), significant expression levels in the fetal brain and other tissues. The study revealed bilallelic expression of C15orf2 gene in testis but more importantly monoallelic paternal-only expression in the fetal brain. (Karin Buiting et al. 2007). Even more recently, these findings have been proof-stamped by analyzing DNA and RNA from a fetal brain and their corresponding DNAs. DNA profiling of four expressed short tandem repeats(STRs), in the 3' untranslated region showed heterozygozity for all four STRs in fetal brain DNA, whereas in fetal brain RNA only one allele could be detected. At all four STRs studied, the expressed allele was of paternal origin (Fig. 1). This procedure was performed using Western blotting and the protein extracts from the brain were analysed with the use of anti-C15orf2 antibody.These results indicate expression of C15orf2 is maternally-imprinted in fetal brain (Michaela Wawrzik et al.)
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Another piece of research carried out by Karin Buiting et al. was the identification of two novel genes found between NDN and C15orf2; PWRN1 and PWRN2. The genes occur in five copies in a 700-kb region (Fig.1B), but only one copy each appears to be expressed. The genes have no protein-coding potential and are subject to alternative splicing and polyadenylation. For PWRN2, expression, which was only detected in testis RNA, was observed to be from both parental alleles (biallelic) concluding that PWRN2 is a non-imprinted gene. On the other hand, despite expression of PWRN1 being most abundant in testis, low levels of expression were also detected in different part of the fetal brain. Analysis by single nucleotide polymorphism (SNP) analysis showed biallelic expression in testis and monoallelic expression in fetal brain. Furthermore we also found four duplicated copies of a CpG island, one of which (termed CpG1) is located 15 kb upstream of exon 1 of PWRN1. Methylation analysis of CpG1 showed complete absence of methylation in DNA from human spermatozoa, but an equal proportion of methylated and unmethylated alleles in DNA from human fetal brain. Due to the absence of any polymorphism close to or inside CpG1 we were not able to find out whether methylation in fetal brain is allele specific. However, monoallelic expression in this tissue suggests that expression is regulated by differential DNA methylation. It can therefore be concluded that PWRN1 is an imprinted gene. Despite the fact that the C15orf2 gene has been discovered for nearly a decade, not a lot of research has been made about its implications in the PWS region. This is mainly due to the fact that there is no equivalent of the C15orf2 gene in mice therefore restricting the number of convenient prototypes for biomedical research.
Partial str profile
One of the most common symptoms of PWS is defects in sexual development linked to hypogonadotrophic hypogonadism (Burman P et al. 2001), which involves the GnRH system.
Gonadotrophin releasing hormone (GnRH) is an essential regulator of the HPG axis which consists of the hypothalamus, pituitary gland and gonads, each endocrine gland working together in several important systems such as the reproductive and immune systems. GnRH is also responsible for the release of FSH (Follicle stimulating hormone) and LH (luteinizing hormone) which are critical for reproductive function.
In this instance, mice were studied as its chromosome region 7c is homologous to the 15q11-q13 region is humans and as a result it has been found that high levels of Necdin, a MAGE family protein coded by the NDN gene, are present in mature GnRH neurons (GT1-7) but no expression/little expression was detected in immature GnRH neurons (GN11) (Nichol Miller at al. 2008). Previous studies also showed that the GnRH gene is regulated by Msx homeodomain repressor proteins, which are expressed in GnRH neurons. Msx transcription factors bind the characterized rat GnRH enhancer and promoter at two ATTA sites in each region, to regulate transcriptional activity of the GnRH gene. Necdin inhibits repression of GnRH gene expression by Msx1 through Msx-binding sites in the GnRH enhancer and promoter regions (Givens ML et al. 2005). Another finding was that Necdin-null mice had fewer GnRH neurons during development migration showing that Necdin not only controls expression of GnRH but also GnRH neuronal development.
Based on the above information it is highly likely that the lack of Necdin plays a significant role in contributing to the hypogonatrophic hypogonadism and infertility symptoms found in PWS patients.
Another example of a MAGE family protein is MAGEL2 encoded by the Magel2 gene located in humans at 41 kb distal to NDN in the PWS region. Research was performed on the function of this gene and the consequences of its absence in mice. The overall results of the experiments showed that the loss of Magel2 led to a reduction in fertility in both sexes and also a decline in early reproductive development. In female mice, vaginal opening and age at which they are sexually receptive were examined to indicate puberty. And it was observed that Magel2-null female mice showed a delayed initiation and duration of puberty. Furthermore, by comparing breeding rates between Magel2-null mice (ranging from 7-24 weeks of age) mating with normal mice and control mice mating with normal mice, it was observed that the former showed a lower number of successful pregnancies, indicating reduced fertility with increase in age. No pregnancies were found when Magel2-null males, aged over 24 weeks, were mated with normal mice, indicating absolute infertility.
Magel2-null mice also showed reduced levels of testosterone in their testes but normal levels of Follicle-stimulating hormone (FSH) and Luteinizing hormone (LH). This showed that the absence of Magel2 does not have any effect on the number of GnRH neurons as opposed to NDN. Comparing testes of control and Magel2-null mice showed no difference in histological features and sperm cells obtained were normal, indicating that despite a reduced testosterone level, function of the male reproductive system is unaltered. Magel-null females were found to have irregular estrous cycles that deteriorate with age. In normal mice estrous cycles are usually around 4-5 days but in Magel-null mice the cycling pattern was considerably extended. Based on the above information it would be fair to say that Magel2 contributes to the reproductive impairment symptom found in PWS patients.
There is another category of small RNA molecules called Small nucleolar RNAs (snoRNAs). They help aid chemical changes of RNAs such as transfer RNAs, ribosomal RNAs and small nuclear RNAs. There are 2 key classes of snoRNA:
The C/D box snoRNAs- these are associated with 2'-O-methylation whereby a methyl group is attached to the 2' hydroxyl group of the ribose moiety of a nucleoside
The H/ACA box snoRNAs- (It should be noted though that no methylation target has been found for HBII-85). The group of snoRNAs that appear to be strongly involved in the pathogenesis of PWS is the C/D box snoRNAs in the PWS region of chromosome 15.
The snoRNAs arise from introns spliced out of paternally expressed RNA transcripts that extend for several hundred kilobases from the bicistronic protein-coding SNURF-SNRPN gene to the UBE3A.There are two snoRNA clusters-HBII-85, with 29 snoRNAs, and HBII-52, with 42 snoRNAs as well as two copies of HBII-438 and several single-copy snoRNAs. In 1991, Hamabe et al. described a Japanese family with a submicroscopic deletion in 15q11-q13. Three siblings, who had the deletion on the maternal chromosome, had Angelman syndrome (AS). The mother had the deletion on the paternal chromosome and was described as being healthy. The deletion breakpoints were cloned and sequenced by Greger et al. in 1993. Based on the DNA sequence information (NCBI accession no. L15422), we have found that the proximal deletion breakpoint is in intron 62 of the SNURF-SNRPN gene. Thus, the deletion includes not only UBE3A, but also all of the 47 HBII-52 genes, which are located between exons 63 and 144 of SNURF-SNRPN (Runte et al. 2001). Gallagher et al. conducted research on the HBII-52 gene and concluded that loss of this particular gene did result in any clinical phenotype and is not involved in the etiology of PWS. However, the presence, in HBII-52, of an 18-bp antisense box complementary to a critical segment of the serotonin 2C receptor mRNA makes these snoRNAs good functional candidates for a role in PWS (Cavaille´ et al. 2000). It has been proved to significantly affect the expression of 5HT2c receptor isoforms in patients with PWS so there is a likelihood that it could contribute indirectly to the phenotype of PWS. We also cannot exclude the possibility that loss of HBII-52 has a direct phenotypic effect when accompanied by the loss of function of other genes in 15q11-q13.
Here, we describe the characterization of a de novo microdeletion in an individual meeting the criteria for a diagnosis of PWS, showing all of seven major revised clinical criteria including neonatal hypotonia, feeding difficulties and failure to thrive during infancy, excessive weight gain after 18 months, hyperphagia, hypogonadism, global developmental delay and equivocal facial features. Additional minor features include behavioural problems, sleep apnea, skin picking, speech delay, and small hands and feet relative to height. This individual was found to have a deletion in the snoRNA region at 15q11.2. Array-based comparative genomic hybridization (array CGH) using a BAC array showed a loss of copy number for two clones encompassing approx. 400 kb within the 15q11-q13 PWS and Angelman Syndrome critical interval. To rule out deletion or imprinting abnormalities causing PWS, we carried out DNA methylation analysis of the PWS-imprinting centre and found a normal methylation pattern. Chromosome analysis showed a normal male karyotype. A combination of high-resolution oligonucleotide-based array CGH and quantitative real-time PCR helped to define the deletion more precisely, and predicted the deletion boundaries to be between positions 22.83 Mb (centromeric) and 23.01 Mb (telomeric). The clinical array used to make the initial diagnosis in the affected individual included 1,475 BAC clones and did not detect any other abnormalities. We identified a junction fragment of B2.6 kb using long-range PCR and breakpoints at position 22,835,594 (proximal) and 23,010,179 (distal) with an insertion of 8 bp using sequencing; thus, the deleted segment was exactly 174,584 bp. The proximal breakpoint occurred between EST AB061718 and snoRNA HBII-438A, and the distal breakpoint was between snoRNA box 23 and 25 of the HBII-52 cluster (Fig. 2b). The deletion encompasses HBII-438A, all 29 sno-RNAs comprising of the HBII-85 cluster, and the proximal 23 of the 42 snoRNAs comprising the HBII-52 cluster. The parental origin of the deletion was confirmed by a polymorphic dinucleotide repeat within the deleted interval; this showed that only a maternal allele was present in the proband, thus placing the deletion on the paternal chromosome. Our data thus reveal a unique microdeletion encompassing the entire HB-II-85 cluster, HBII-438A, and a portion of the HB-II-52 cluster of snoRNAs. Our data is now notably in favor of the interpretation that paternal deficiency of HBII-85 causes the key manifestations of the PWS phenotype, although some atypical features suggest that other genes in the region may make lesser phenotypic contributions.
(include diagram from sahoo et al)
The SNURF-SNRPN gene found on chromosome 15 seems to be one of the most complex loci within the human genome. It consists of 10 functional exons, transcribing into a 1.4Kb bicistronic transcript. Ozcelik et al. researched the function of these exons and found that the SNURF protein is encoded by exons 1-3 and the SmN splicosomal protein is encoded by exons 4-10. It also contains several untranslated exons whose functions remain to be established. In addition, this locus also contains the PWS IC element, spanning exon 1 and which is essential for parental imprinting. Wirth J. Back et al. detected an additional 8 non-coding 3'exons of the SNURF-SNRPN gene. Maren et al. identified two extra functions of the SNURF-SNRPN gene, firstly that it serves as a host for multiple small nucleolar RNA species and secondly it is the start site for the UBE3A antisense transcript.