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GENOME IMPRINTING – Ayesha Tungekar

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GENOME IMPRINTING

 

Ayesha Tungekar

 

GENOME IMPRINTING DISORDERS:

 

PRADER WILLI SYNDROME

 

Prader Willi syndrome PWS) is caused due to mutation on parental chromosome 15(15q11-q13 region) which results in deletion of certain important gene. 15q11-q13 contains paternally expressed SNRP gene and maternally expressed UBE3A gene. There is a loss of paternal expression in the consensus sequence. In 70% of cases, PWS is caused due to non-inherited deletion in paternally derived chromosome 15 region. In 25% of cases, PWS results from maternal disomy. Both chromosome 15 are inherited from mother.In 2% of cases translocation occurs (i.e., rare chromosomal rearrangement)that causes the gene to switch off paternal chromosome.  (Butler, 2009)

Image1: Prader Willi syndrome

                                                  Image1: Prader Willi syndrome

                            Source-sgugenetics / Inheritance of PWS and AS (pbworks.com)

     The typical PWS deletion consists of two classes, Type I and Type II.

  1. Type I deletion involves breakpoint 1 (BP1) which is nearest the centromere. It is the Larger deletion

  2. Type II deletion involves breakpoint 2 (BP2). It is a smaller deletion.

  3. Breakpoint 3 (BP3) is common in both typical deletion subgroups.

SYMPTOMS OF PRADER WILLI SYNDROME:

Symptoms are caused due to delayed function of the brain. Hypotonia, weak cry, temperature instability underdeveloped sex organs are generally seen in an infant. Instable appetite, rapid weight gain, continued developmental delay, psychomotor retardation, decrease pain sensitivity is seen in children of 2- 4 years of age. Prader-Willi syndrome patients have fair skin and light-colored hair if there is mutation in OCA2 gene of chromosome 15.

ANGELMAN SYNDROME.

Angelman syndrome (AS) is caused by single imprinted maternally expressed gene (i.e., UBE3A gene). Angelman syndrome (AS) also involves mutation on chromosome 15q but in a different specific region.

IN 70% of cases AS syndrome occurs when the segment of chromosome 15q containing the UBE3A gene is deleted. UBE3A,  a ubiquitin ligase gene is involved in the ubiquitin pathway, early brain development and therefore removal f this gene leads to deficiencies in the brain. 11% of cases arise due to mutation in the maternal copy of the UBE3A gene. In 6% of cases AS syndrome is caused due inheritance of two copies of chromosome 15 from father (paternal copies) instead of one copy from each parent. (Butler, 2009)

Angelman Syndrome                           

                                                Image 2: Angelman Syndrome

                          Source- https://chelseausher.files.wordpress.com/2012/04/as1.jpg

 SYMPTOMS OF ANGELMAN SYNDROME

AS is characterized by seizures, lack of speech, intellectual disability, largemouth with protruding tongue, hypopigmentation, happy excitable appearance with frequent laughter and smiling, most of the children face difficulty in sleeping.

GENETIC ANALYSIS OF PWS AND AS SYNDROME:

Prader Willi and Angleman Syndrome are distinct neurological disorders. These disorders are caused due to genetic alterations in chromosome 15q11-13. It is difficult to diagnose these syndromes solely based on their clinical symptoms as they have clinical overlaps with other disease and therefore genetic testing needs to performed.

Collect blood samples from patients having hypotonia, showing d delayed psychomotor development, weak cry. Isolate the genomic DNA by using genomic DNA isolation kits

  • Methylation-specific PCR.

MS-PCR helps in the analysis of DNA methylation patterns OF CpG islands. Methylation is an important epigenetic phenomenon which plays important role in genome imprinting. Genomic DNA needs to be treated by using Bisulfite Modification Kit. The CpG dinucleotides are methylated on maternal chromosome and unmethylated on the paternal chromosome. When primer set that are complementary to the sequence with methylated CpGs, but are not complementary to the originally same sequence with unmethylated CpGs are used for PCR, only the sequence (allele) with methylated CpGs should be amplified. The same is true for the primer pair specific for the sequence with unmethylated CpGs.The methylated SNRPN locus was amplified by using MF 5’-TAAATAAGTACGTTTGCGCGGT-3′ and MR 5’-AACCTTACCCGCTCCATCG-3′ primers which generated 174base pair methylation product and non-methylated allele can be amplified using PF 5′-GGTTGGTGTGTATGT TTAGGT-3′ and PR 5’-TCAAACATCTCCAACA ACCA-3′ which generates around 100base pair product. (Liu et al., 2019)

 

 

MS-PCR analysis of SNRPN gene in PWS/AS patient

                                Image3:MS-PCR analysis of SNRPN gene in PWS/AS patient

          Source- Liu et al. Molecular Cytogenetics (2019) 12:7 https://doi.org/10.1186/s13039-019-0420

  • STR analysis

Makes use of STR (short tandem repeat) markers. STR (also called microsatellites) are polymorphic DNA used in linkage mapping studies, to identify the underlying genetic mechanism. These STR markers can be used for analysis to understand paternal/maternal deletions and uniparental disomy.  Multiplex fluorescence-based STR analysis can identify molecular defects between a typical deletion and UPD i.e. we can distinguish whether the syndrome is caused due to deletion on chromosome q5 or due to inheritance of two copies of chromosome 15. (Liu et al., 2019)

Collect blood samples from patients having hypotonia, showing d delayed psychomotor development, weak cry. Isolate the genomic DNA by using genomic DNA isolation kits

  • Methylation-specific PCR.

MS-PCR helps in the analysis of DNA methylation patterns OF CpG islands. Methylation is an important epigenetic phenomenon which plays important role in genome imprinting. Genomic DNA needs to be treated by using Bisulfite Modification Kit. The CpG dinucleotides are methylated on maternal chromosome and unmethylated on the paternal chromosome. When primer set that are complementary to the sequence with methylated CpGs, but are not complementary to the originally same sequence with unmethylated CpGs are used for PCR, only the sequence (allele) with methylated CpGs should be amplified. The same is true for the primer pair specific for the sequence with unmethylated CpGs.The methylated SNRPN locus was amplified by using MF 5’-TAAATAAGTACGTTTGCGCGGT-3′ and MR 5’-AACCTTACCCGCTCCATCG-3′ primers which generated 174base pair methylation product and non-methylated allele can be amplified using PF 5′-GGTTGGTGTGTATGT TTAGGT-3′ and PR 5’-TCAAACATCTCCAACA ACCA-3′ which generates around 100base pair product. (Liu et al., 2019)

 

MS-PCR analysis of SNRPN gene in PWS/AS patient

MS-PCR analysis of SNRPN gene in PWS/AS patient

                                Image3:MS-PCR analysis of SNRPN gene in PWS/AS patient

          Source- Liu et al. Molecular Cytogenetics (2019) 12:7 https://doi.org/10.1186/s13039-019-0420

  • STR analysis

Makes use of STR (short tandem repeat) markers. STR (also called microsatellites) are polymorphic DNA used in linkage mapping studies, to identify the underlying genetic mechanism. These STR markers can be used for analysis to understand paternal/maternal deletions and uniparental disomy.  Multiplex fluorescence-based STR analysis can identify molecular defects between a typical deletion and UPD i.e. we can distinguish whether the syndrome is caused due to deletion on chromosome q5 or due to inheritance of two copies of chromosome 15. (Liu et al., 2019)

  • Chromosomal microarray analysis.

CMA analysis is a comprehensive genome testing that helps to detect a genetic condition responsible for causing the disability. CMA analysis helps to identify breakpoints (BPI, BP2, BP3) in confirmed cases of PWS and AS syndrome patients. (Liu et al., 2019)

CMA results of patients

            Image 4-  CMA results of patients with PWS/AS. Source: (Liu et al., 2019)

 

GENOME IMPRINTING AND TUMORIGENESIS.

During gametogenesis the alleles appears to be differentially marked which is heritable but reversible from generation to generation implying a stable epigenetic modification. Dysregulation of normal imprinted gene leads to tumor development. We know that Genomic imprinting is vital for normal development, it is the phenomenon in which individual alleles of certain gene are expressed differentially according to parent of origin. Beckwith–Wiedemann syndrome (BWS). This syndrome is characterized by increased risk of childhood tumor. Abnormal methylation patterns is seen in H19 gene (maternally expressed) and IGF-2 gene (paternally expressed) in chromosome 11p15.The maternally expressed H19 gene encodes a polyadenylated-spliced

message and is assumed to act as a growth-suppressing agent In this syndrome there is abnormal expression of insulin-like growth factor 2 gene due to loss of imprinting . Loss of imprinting causes breakdown in normal suppression by maternal allele which increases IGF2 expression. (Joyce & Schofield, 1998)

 

 

 

REFERENCES:

 

  1. Alan M. O’DohertyDavid E. MacHughCharles Spillane and David A. Magee (2015), Genomic imprinting effects on complex traits in domesticated animal species, frontiers of genetics.

  1. Butler, M. G. (2009). Genomic imprinting disorders in humans: A mini-review. Journal of Assisted Reproduction and Genetics, 26(9–10), 477–486. https://doi.org/10.1007/s10815-009-9353-3

  1. Jinno, Y. (1996). Genomic imprinting. Journal of Human Genetics, 41(1), 19. https://doi.org/10.1097/mop.0000000000000072

  1. Joyce, J. A., & Schofield, P. N. (1998). Genomic imprinting and cancer. Molecular Pathology, 51(4), 185–190. https://doi.org/10.1136/mp.51.4.185

  1. Liu, C., Zhang, X., Wang, J., Zhang, Y., Wang, A., Lu, J., Huang, Y., Liu, S., Wu, J., Du, L., Yang, J., Ding, H., Liu, L., Zhao, X., & Yin, A. (2019). Genetic testing for Prader-Willi syndrome and Angelman syndrome in the clinical practice of Guangdong Province, China. Molecular Cytogenetics, 12(1), 7.

  1. Simmons, D. (2008)Epigenetic influence and disease. Nature Education 1(1):6

  1. Tucci, V., Isles, A. R., Kelsey, G., Ferguson-Smith, A. C., Bartolomei, M. S., Benvenisty, N., … Wilkins, J. (2019). Genomic Imprinting and Physiological Processes in Mammals. Cell, 176(5), 952–965. https://doi.org/10.1016/j.cell.2019.01.043

  2. Yufeng Li & Hiroyuki Sasaki(2011)Genomic imprinting in mammals: its life cycle, molecular mechanisms and reprogramming, Cell Research, 21, pages:466–473.

  1. William A. MacDonald(2011)Epigenetic Mechanisms of Genomic Imprinting: Common Themes in the Regulation of Imprinted Regions in Mammals, Plants, and Insects, Genetics Research International Volume 2012

 

Ayesha Tungekar 
Research writer

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