Genomic Imprinting and Uniparental Disomy in Medicine

REFERENCES 11

Nicholls, R. D., Saitoh, S. and Horsthemke, B. Imprinting in Prader-Willi and Angelman syndromes. Trends Genet 14:194–200, 1998.

Purvis-Smith, S. G., Saville, T., Manass, S., et al. Uniparental disomy 15 resulting from ‘‘correction’’ of an initial trisomy 15 [letter]. Am J Hum Genet 50:1348–1350, 1992.

Reik, W., Collick, A., Norris, M. L., Barton, S. C. and Surani, M. A. Genomic imprinting determines methylation of parental alleles in transgenic mice. Nature 328:248–251, 1987.

Rousseau-Merck, M. F., Versteege, I., Legrand, I., et al. hSNF5=INI1 inactivation is mainly associated with homozygous deletions and mitotic recombinations in rhabdoid tumors. Cancer Res 59:352–3156, 1999.

Sapienza, C. Genome imprinting and dominance modification. Ann N Y Acad Sci 564:24–38, 1989.

Sapienza, C. Parental imprinting of genes. Sci Am 263:52–60, 1990.

Searle, A. G. and Beechey, C. V. Complementation studies with mouse translocations.

Cytogenet Cell Genet 20:282–303, 1978.

Searle, A. G. and Beechey, C. V. Non-complementation phenomena and their bearing on nondisjunctional effects. In: Aneuploidy: etiology and mechanisms, edited by Dellarco, V., Voytek, P. and Hollander, P. 1985,

Snell, G. D. An analysis of translocations in the mouse. Genetics 31:157–180, 1946.

Solter, D. Differential imprinting and expression of maternal and paternal genomes. Annu Rev Genet 22:127–146, 1988.

Spence, J. E., Perciaccante, R. G., Greig, G. M., et al. Uniparental disomy as a mechanism for human genetic disease. Am J Hum Genet 42:217–226, 1988.

Stephens, J. D. Karyotypes of 1142 couples with recurrent abortion [letter; comment]. Obstet Gynecol 73:144–145, 1989.

Sulisalo, T., Francomano, C. A., Sistonen, P., et al. High-resolution genetic mapping of the cartilage-hair hypoplasia (CHH) gene in Amish and Finnish families. Genomics 20:347– 353, 1994.

Surani, M. A. Genomic imprinting: developmental significance and molecular mechanism.

Curr Opin Genet Dev 1:241–246, 1991.

Surani, M. A., Barton, S. C. and Norris, M. L. Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature 308:548–550, 1984.

Surani, M. A., Kothary, R., Allen, N. D., et al. Genome imprinting and development in the mouse. Dev Suppl 89–98, 1990.

Takagi, N. and Sasaki, M. Preferential inactivation of the paternally derived X chromosome in the extraembryonic membranes of the mouse. Nature 256:640–642, 1975.

Temple, I. K., Gardner, R. J., Robinson, D. O., et al. Further evidence for an imprinted gene for neonatal diabetes localised to chromosome 6q22-q23. Hum Mol Genet 5:1117–1121, 1996.

Vaughan, J., Ali, Z., Bower, S., Bennett, P., Chard, T. and Moore, G. Human maternal uniparental disomy for chromosome 16 and fetal development. Prenat Diagn 14:751–756, 1994.

Vidaud, D., Vidaud, M., Plassa, F., Gazengel, C., Noel, B. and Goossens, M. Father to son transmission of hemophilia A due to uniparental disomy. Am J Hum Genet 59:A2261989.(Abstract)

12 INTRODUCTION

Voss, R., Ben-Simon, E., Avital, A., et al. Isodisomy of chromosome 7 in a patient with cystic fibrosis: could uniparental disomy be common in humans? Am J Hum Genet 45:373–380, 1989.

Wang, J. C., Passage, M. B., Yen, P. H., Shapiro, L. J. and Mohandas, T. K. Uniparental heterodisomy for chromosome 14 in a phenotypically abnormal familial balanced 13=14 Robertsonian translocation carrier. Am J Hum Genet 48:1069–1074, 1991.

Genomic Imprinting and Uniparental Disomy in Medicine: Clinical and Molecular Aspects

Eric Engel, Stylianos E. Antonarkis

Copyright # 2002 Wiley-Liss, Inc. ISBNs: 0-471-35126-1 (Hardback); 0-471-22193-7 (Electronic)

Chapter 2

Detection of Uniparental Disomy and Imprinting by DNA Analysis

In this chapter, we briefly discuss the different laboratory methods used for the detection of uniparental disomy (UPD) and imprinting (or parent-of-origin allelic expression). Only the common and widely used methods are discussed; for an exhaustive and detailed analysis, the reader is referred to the numerous articles in the relevant literature.

DETECTION OF UNIPARENTAL DISOMY IN DNA

The presence of two copies of a locus originating from one parent only and the absence of contribution from the other can be best documented by the use of DNA polymorphisms. For this analysis, DNA from both parents and the proband is needed in order to determine the parental origin of each chromosome. The necessary DNA polymorphisms, which could serve as molecular markers to distinguish the alleles of a locus in a given chromosome, are heritable and abundant variations of the DNA sequence. There exist several types of DNA polymorphisms.

(i) Single nucleotide polymorphisms (SNP). An average 1 in 600–1300 nucleotides varies between two randomly chosen chromosomes (Kan and Dozy, 1978; Antonarakis et al., 1982; Chakravarti et al., 1984; Nickerson et al., 1998; Halushka et al., 1999; Cargill et al., 1999; Venter et al., 2001; Sachidanandam et al., 2001). Millions of SNPs exist in the human genome and could be detected by several methods (Dean, 1995). The sequence of the human genome revealed

13

14 DETECTION OF UNIPARENTAL DISOMY AND IMPRINTING BY DNA ANALYSIS

more than a million of potential SNPs. (Venter et al., 2001; Sachidanandam et al., 2001). A frequently updated database of SNPs has been established at <http:==www.ncbi.nlm.nih.gov=SNP=>. These are usually biallelic polymorphisms, i.e., there exist two different alleles in the population. Their usefulness depends on the allele frequency, or heterozygote frequency (Botstein et al., 1980).

(ii) Variable number of short sequence repeats (SSR) or microsatellites. The repeat unit is usually two to five nucleotides. The most abundant of these are the dinucleotide repeat polymorphisms, but those due to tri-tetra- or penta-nucleotide repeats are much easier for allele scoring (Litt and Luty, 1989; Weber and May, 1989; Tautz, 1989). The repeat unit could also be a mononucleotide run (Economou et al., 1990). The main advantage of the SSR polymorphisms is that there usually exist more than two alleles per locus and therefore the informativeness of SSRs is higher than that of SNPs. There is usually one polymorphic SSR for every 10–50 Kb of human genomic DNA. A public database for SSRs is available at <http:==gdbwww.gdb.org=>. The use of these polymorphic markers was greatly facilitated after the discovery of the polymerase chain reaction (Saiki et al., 1985).

(iia) Variable number of longer repeats (formerly called VNTR for variable number of tandem repeats) in which the repeat unit is usually 20–60 nucleotides (Wyman and White, 1980; Jeffreys et al., 1985; Nakamura et al., 1987). These are also highly polymorphic with many alleles at a given locus, but not as common as the SSRs and not easily detectable by PCR.

(iii) Presence or absence of retrotransposons such as Alu and LINE sequences (long interspersed repeat element) or processed pseudogenes (Schuler et al., 1983; Anagnou et al., 1984; Woods-Samuels et al., 1989). These polymorphisms also comprise diallelic systems, (i.e., there are only two alleles per locus) and are easy to detect, but much rarer than the previous categories.

The basis for the detection of UPD using DNA polymorphisms is shown schematically in the example of Figure 1. In the pedigree of this figure, the DNA of the father (individual 1) is heterozygous for two different polymorphic alleles A and B at locus X. Mother’s DNA (individual 2) contains two yet different alleles C and D of locus X (this is obviously an SSR polymorphic locus with many alleles). The second male offspring of these parents (individual 4) inherited alleles A and B from the father; no maternally inherited alleles at this locus are present. The diagnosis of paternal uniparental disomy (UPD) is obvious (assuming that the probability of de novo mutation of one of the maternal alleles to A or B is negligible and there is no laboratory error). In this example, we observe heterodisomy for locus X since both paternal alleles are present in the DNA of individual 4. In the case of the third child (individual 5), there is homozygosity of allele B of locus X and we therefore observe isodisomy for locus X. Because of the meiotic recombination, it is not unusual to detect isodisomy for one portion of the chromosome and heterodisomy for another. It is also possible to detect UPD for only one segment of the chromosome and normal biparental inheritance for the remainder. In summary, the use of DNA markers allows the determination of the inheritance of specific parental alleles and the detection of the absence of parental alleles in the offspring.

DETECTION OF DELETIONS BY DNA POLYMORPHISMS ANALYSIS

15

Figure 1 Schematic representation of the use of DNA polymorphisms in determining the inheritance of parental alleles. A, B, C, and D depict the four different alleles of locus X on the long arm of the chromosomal pair shown next to the symbol of each individual. An image of the polymorphic alleles after gel electrophoresis and autoradiography is shown on the right panel. For the interpretation of the results of the analysis, see the text.

The detection of the polymorphic alleles of a given locus can be performed using numerous laboratory techniques (Dean, 1995). Almost all use the polymerase chain reaction amplification (Saiki et al., 1985), and subsequently the detection is performed by restriction endonuclease analysis (Kan and Dozy, 1978), allele-specific oligonucleotide hybridization (Conner et al., 1983; Chee et al., 1996), allele-specific amplification (Newton et al., 1989; Wu et al., 1989), single-stranded conformation analysis (Orita et al., 1989), enzymatic or chemical cleavage of nucleotide mismatches (Cotton et al., 1988; Youil et al., 1995), denaturing gradient gel electrophoresis (Myers et al., 1985), denaturing high-pressure liquid chromatography (Underhill et al., 1997), sequence analysis (Sanger et al., 1977); all for SNPs. For the SSRs, the method used is PCR and acrylamide gel electrophoresis for fragment size discrimination that could be done either manually or using the automated nucleotide sequencers.

DETECTION OF DELETIONS BY DNA POLYMORPHISMS ANALYSIS

The detection of a de novo (micro)deletion in DNA and its parental origin could be carried out by the use of informative DNA polymorphisms in the DNAs of the proband and its parents, as shown schematically in Figure 2. In the case of a

16 DETECTION OF UNIPARENTAL DISOMY AND IMPRINTING BY DNA ANALYSIS

Figure 2 Schematic representation of the detection of DNA deletions using polymorphisms. In the family on the left, the child inherited a paternal chromosome with a de novo deletion. The polymorphism used maps within the deletion and there is therefore no paternal allele seen in the child’s DNA. This situation is common in PWS. In contrast, in the family on the right, the child inherited a maternal chromosome with a de novo deletion, and consequently, there is no maternal allele in the DNA of the child.

paternally derived deletion, there is no detectable paternal allele transmitted to the proband; similarly, in the case of a maternally derived deletion, no maternal allele is seen in the DNA of the proband. The limitation of this method is that the polymorphisms used need to be informative in a given family, i.e., there are allelic differences in the parents that permit the detection of the absence of parental contribution. In addition, in the case of parentally derived deletion, nonpaternity needs to be excluded with the use of numerous other polymorphic loci in various chromosomes. Finally, this analysis requires the availability of DNA samples from both parents of the proband, which is not always feasible.

DETECTION OF PARENT-OF-ORIGIN ALLELE-SPECIFIC EXPRESSION

The phenomenon of imprinting can be defined as the specific expression of only one parental allele, but not both, in a specific tissue. For example, expression of gene X is found in blood only from the paternal allele, although a maternal allele is also present in the DNA of the individual.

DETECTION OF PARENT-OF-ORIGIN ALLELE-SPECIFIC EXPRESSION

17

The polymorphic variability in the coding region and 50 UTR and 30 UTR (untranslated region) of human genes provides ample opportunities for the detection of allele-specific expression. It has been recently shown that there are abundant SNPs in the RNA products of human genes (Cargill et al., 1999; Halushka et al., 1999). The basic principle of the detection of the allele-specific expression is to mark the transcript from each allele with a different polymorphism so that the parent-of- origin specific expression could be determined (Rainier et al., 1993). In the example of Figure 3, the paternal gene is marked with an SNP that could be digested with the restriction enzyme EcoRI, whereas the maternal gene contains the SNP allele that is noncleavable by this enzyme. A reverse transcriptase-PCR (RT-PCR) fragment that

	E
5’	3’	1		2	5’	3’
5’	3’
					5’	3’
	E
		3		4
	3	4	3	4
	E
5’	3’				Maternal
5’	3’
OL1	OL2 OL3				Paternal
	DNA			RNA
	PCR		RTPCR
	( OL2 - OL3 )		( OL1 - OL3 )

Figure 3 Schematic representation of the detection of differential gene expression related to the parental origin of the gene. In this example, the paternally derived allele of the gene contains a single nucleotide polymorphism (SNP) that creates a recognition site for the restriction enzyme EcoRI, whereas the maternally derived allele lacks this recognition site. Reverse transcription PCR using oligonucleotide primers OL1 and OL3 results in amplification only from RNA, while PCR using oligonucleotide primers OL2 and OL3 is used to amplify fragments from genomic DNA. Digestion of the RT-PCR products with EcoRI detects transcripts from only the paternal allele since all these amplification products contain the EcoRI restriction site. Analysis of the genomic DNA shows that both the paternal and maternal alleles are present. In this example, the maternal allele is silent, and only the paternal allele is expressed. In this kind of analysis, the investigator should always eliminate the possibility of a deleterious mutation in the nonexpressed allele.

18 DETECTION OF UNIPARENTAL DISOMY AND IMPRINTING BY DNA ANALYSIS

contains the EcoRI SNP could then be used to discriminate the expression of the two alleles. The oligonucleotide primers for the RT-PCR are placed in different exons, so that the specific amplification originates only from the RNA template and not the genomic DNA. In the example of Figure 3, RT-PCR amplification of only fragments containing the EcoRI site (cleavable) establishes expression of the paternal allele only, whereas RT-PCR amplification of the fragments that do not contain the EcoRI site (noncleavable) fragments documents the expression of the maternal allele only. RT-PCR amplification from both alleles is the common situation of biallelic expression. As with the previous example of detection of uniparental disomy, the existence of the normal variability in the genome provides the tools for the detection of differential allelic expression.

DETECTION OF DIFFERENTIAL METHYLATION BY RESTRICTION ANALYSIS

The parent-of-origin differential allele expression is often associated with differential methylation of cytosine in CpG dinucleotides. Overmethylation has been associated with inactive genes and undermethylation with active, expressed genes (Cedar, 1988). It has been therefore useful to use the detection of differential methylation as a tool for diagnosis of differential allelic expression. The methylation detection is routinely used in the diagnosis of Prader-Willi and Angelman syndromes (see Chapters 6 and 7) (Glenn et al., 1993). In the normal situation, the methylation status of both parental loci on the chromosomal region 15q11-q13 is observed. In PraderWilli syndrome, only the maternal locus with its specific methylation status is observed; in contrast, in Angelman syndrome only the paternal locus with its specific methylation status is seen. This is shown schematically in Figure 4, in which the differential methylation status is assessed by the use of the methylation-sensitive restriction enzyme HpaII that cleaves its recognition DNA sequence 50 CCGG30 when it contains unmethylated cytosine, while it does not cleave when its recognition sequence contains a methylated C. In the example of Figure 4, the discrimination between the methylated (maternal) and the unmethylated (paternal) allele is performed after digestion of the DNA with EcoRI and HpaII, Southern blotting of the resulting fragments, and hybridization with a specific probe as shown. The HpaII digestion is only successful in the unmethylated allele, resulting in a small fragment; in contrast, the failure of HpaII to cleave the methylated site results in the large fragment in the Southern blot analysis. In the Prader-Willi syndrome, only the methylated, ‘‘larger’’ maternal fragment is observed; conversely in the Angelman syndrome, only the unmethylated ‘‘smaller,’’ paternal fragment seen by the probe is observed. This study of selected methylated sites within the 15q11-q13 region is a useful diagnostic procedure that is now routinely used in molecular diagnostic laboratories throughout the world (ASHG=ACMG, 1996).

PCR DETECTION OF METHYLATION DIFFERENCES

19

	Normal		PWS		AS
E	H			E	H
	E				E
PAT	CG				CG
	E	E	E
E					probe
	CG		CG
MAT
	probe		probe
	H		H
			H		H
				E
		E	E		E
			CG		CG
Normal PWS AS		E	E	E	E
			CG		CG
	Maternal		probe		H probe
			H

Paternal

EcoRI and HpaII

digestion

Figure 4 Schematic representation of the detection of methylation differences using restriction endonuclease analysis. The paternally derived allele is non methylated at the CpG dinucleotide shown between two EcoRI (E) restriction sites and therefore this site is cleaved by HpaII (H) endonuclease. In contrast, the maternally derived allele is methylated at this CpG site (shown by the gray circle) and therefore not cleaved by HpaII. The probe for the Southern blot analysis is designated with a bracket. The methylated (maternal) allele is detected as a large band after autoradiography, while the unmethylated (paternal) allele results in a smaller EcoRI-HpaII fragment. In the PWS, the methylation pattern is only maternal due to either a deletion of the paternal allele (top panel) or the abnormal, maternal-type methylation status of the paternal allele. In contrast, in some cases of the AS, the methylation pattern is only paternal due to either a deletion of the maternal allele (top panel) or the abnormal, paternal-type methylation status of the maternal allele.

PCR DETECTION OF METHYLATION DIFFERENCES

The differences of allele-specific methylation can also be detected by PCR analysis without Southern blotting, although this method is not widely used in routine diagnostic evaluations. The basis of the technique is that DNA treated with sodium bisulfite converts cytosine to uracil except when cytosine is methylated. After this chemical DNA modification, oligonucleotide primers specific for the methylated and unmethylated versions of the CpG clusters are utilized for PCR amplification of specific DNA fragments originating only from the methylated or unmethylated modified templates (Herman et al., 1996; Kubota et al., 1997). The method is shown schematically in Figure 5.

20 DETECTION OF UNIPARENTAL DISOMY AND IMPRINTING BY DNA ANALYSIS

		Normal			PWS					AS
	Sodium Bisulfide
		treatment
	PAT specific PCR				MAT specific PCR			PAT specific PCR
OL1		OL2		OL3		OL4		OL1		OL2

PAT

MAT

UG

UG

UG

UG

CG

CG

CG

CG

UG

UG

UG

UG

CG

CG

CG

CG

CG

CG

CG

CG

UG

UG

UG

UG

OL3		OL4		OL3		OL4		OL1		OL2
	MAT specific PCR				MAT specific PCR				PAT specific PCR

Normal PWS AS

Maternal

Paternal

PCRs with OL1 - OL2 and OL3 - OL4

Figure 5 Schematic representation of the methylation-specific PCR. DNA treated with sodium bisulfite that converts cytosine to uracil except when cytosine is methylated. Oligonucleotide primers OL1 and OL2 were designed to complement ‘‘methylated,’’ i.e., unmodified, cytosines and therefore their PCR product is only maternal-specific since these oligonucleotides do not bind the modified DNA. In contrast, oligonucleotides OL3 and OL4 were designed to complement ‘‘unmethylated,’’ i.e., modified, cytosine to uracil residues. Their PCR product is only paternalspecific since these oligonucleotide primers do not bind the unmodified DNA. The paternal or maternal allele-specific amplification is recognized by the specific size of the PCR amplification product. In PWS patients, a maternal methylation PCR pattern is only observed, either because there is a deletion of the paternally derived allele or an abnormal, maternal-type methylation status of the paternal allele. In AS patients, a paternal methylation PCR pattern is only observed, either because there is a deletion of the maternally derived allele or the abnormal, paternal-type methylation status of the maternal allele.

DETECTION OF (MICRO)DELETIONS BY FISH

The technique of in situ hybridization is based on the principle that, given the appropriate temperature and salt conditions, single-stranded DNA reanneals with its complementary sequence into double-stranded DNA. A single-stranded labeled probe (usually the DNA insert of a cosmid) is allowed to hybridize with the DNA of metaphase chromosomes fixed on a glass slide (Gerhard et al., 1981; Garson et al., 1987). The probe DNA is labeled with nonisotopic compounds that could then be detected using fluorescence or enzymatic linked reactions. Biotin and digoxigenin