Molecular Genetic Findings in Prader-Willi Syndrome 3

3

Molecular Genetic Findings in Prader-Willi Syndrome

Karin Buiting and Bernhard Horsthemke

Prader-Willi syndrome (PWS), the most common genetic cause of marked obesity in humans,

is due to loss of expression of paternal genes from the 15q11–q13 region under the control of an imprinting center. PWS and Angelman syndrome, an entirely different clinical condition due to lack of maternally expressed genes, were the fi rst examples in humans of genomic imprinting. There are three recog- nized genetic subtypes in PWS, including paternally derived interstitial deletions of the 15q11–q13 region, maternal uniparental disomy 15 (both 15 s from the mother), and imprinting defects.

Genomic Imprinting

The chromosomal region 15q11–q13 contains a cluster of genes that are expressed from the paternal or maternal chromosome only (Figure 3.1).

This peculiar expression pattern is a consequence of genomic imprint- ing, which is an epigenetic process by which the paternal and the maternal germ lines mark specifi c chromosome regions. In each gen- eration, the parental imprints are erased and reset according to the sex of the individual (Figure 3.2A). The mechanisms underlying genomic imprinting are not completely understood, but parent-of-origin- specifi c DNA methylation plays an important role in this process. DNA methylation refers to the addition of a methyl group (CH

) to carbon atom 5 of cytosine (Figure 3.2B and C). Only cytosines followed by guanine are methylated. The sequence 5¢-CG-3¢/3¢-GC-5¢ is palindro- mic, and the dinucleotides are either methylated or unmethylated on both strands. De novo DNA methylation is catalyzed by the DNA meth- yltransferases 3A and 3B (DNMT3A and DNMT3B). After DNA repli- cation, the newly synthesized DNA strand is methylated by the maintenance DNA methyltransferase 1 (DNMT1). In general, methyla- tion of the promoter region of a gene means gene silencing.

Within the imprinted region in 15q11–q13, most genes are methyl-

ated and silenced on the maternal chromosome and expressed from the

unmethylated paternal allele only. A paternally derived deletion of this

region, the absence of a paternal chromosome 15 in maternal uniparen- tal disomy 15, or the silencing of the paternal alleles by an imprinting defect lead to a complete loss of function of the paternally expressed 15q11–q13 genes and Prader-Willi syndrome (Figure 3.3). It is still a matter of debate whether PWS is caused by the loss of function of a single gene or of several genes, with growing evidence supporting the involvement of more than one gene.

Deletions and Translocations

A 4 Mb de novo interstitial deletion of the paternal chromosome [del(15)(q11q13)], which includes the entire imprinted domain plus several nonimprinted genes, is found in about 70% of subjects with PWS. The deletion is visible by high resolution chromosome banding analysis and was fi rst described by Ledbetter and colleagues in 1981.

The deletion occurs at a frequency of about 1 in 10,000 newborns and is probably one of the most common deletions observed in humans. In a few patients, the region is deleted as the result of an unbalanced translocation. By studying the inheritance of chromosomal polymor- phisms, Butler and Palmer in 1983

demonstrated that the deletion

Figure 3.1. Schematic overview of human chromosomal region 15q11–q13. White boxes represent genes expressed from the paternal chromosome only; black boxes represent genes expressed from the mater- nal chromosome only; and gray boxes represent genes expressed from both chromosomes. Orientation of transcription, or gene expression, is indicated by horizontal arrows. The snoRNA genes are indicated as grouped or ungrouped vertical lines and the two critical imprinting center (IC) elements for AS (black circle) and PWS (white circle) are shown. Vertical arrows show the positions of translocation breakpoints on chromosome 15 that have been reported for 5 patients with PWS. The breakpoint cluster regions are indicated as black bars. The extension of the class (type) I and class (type) II deletions and the atypical deletions in patients AS deletion 1³⁵ and AS deletion 2¹⁶ are drawn as horizontal lines. The proximal deletion breakpoints of AS deletion 1 and AS deletion 2 defi ne the distal boundary of the PWS critical region.

Figure 3.2. A. Genomic imprinting. During gametogenesis, parental imprints are erased and reset according to the sex of the individual. Imprints are maintained during mitotic cell divisions. White boxes represent paternal imprint; black boxes represent maternal imprint; gray boxes represent no imprint. B. Methylated cytosine. C. Methylation of CG dinucleotides in both DNA strands.

Figure 3.3. Genetic lesions in PWS. White boxes represent genes expressed from the paternal chromo- some only, and black boxes represent genes expressed from the maternal chromosome only. Orientation of transcription, or gene expression, is indicated by vertical arrows.

always occurs on the chromosome 15 inherited from the father. This fi nding was confi rmed in 1989 at the molecular level by Knoll and co- workers,

who also demonstrated that chromosome 15 deletions in Angelman syndrome (AS), which affect the same chromosome region, always occur on the maternal chromosome. It is now known that AS results from the loss of function of the UBE3A gene, which in the brain is expressed from the maternal chromosome only (Figure 3.1).

At the molecular level, two classes or types of deletions (types I and II) can be distinguished. In both types, the distal breakpoints are close to, but telomeric to the P gene (breakpoint region 3, BP3, Figure 3.1).

In type I deletions (30%–40% of patients), the proximal breakpoint is centromeric to the marker D15S541 (breakpoint region 1, BP1). In type II deletions (60%–70% of patients), the proximal break- point is between D15S541 and D15S543 (breakpoint region 2, BP2).

In order to analyze the chromosomal mechanisms underlying deletions in PWS, Carrozzo et al.

and Robinson et al.

genotyped families of PWS and AS patients with the help of microsatellite markers fl anking the common deletion region. Both groups obtained evidence that the deletions can occur by crossover events between the two homologous chromosome 15s (interchromosomal) or between different regions of one chromosome 15 (intrachromosomal). The recurrence risk is very low if the parents have normal chromosomes.

The clustering of the deletion breakpoints in most of the patients with del(15)(q11q13) suggested that there are duplicated sequences that are susceptible to nonhomologous crossovers. Interestingly, proximal 15q is involved in other cytogenetic rearrangements also. For example, inv dup(15) with breakpoints in q11.2 or q13 account for approximately 50% of all supernumerary marker chromosomes.

Less frequent are duplications, triplications, and inversions of this chromosomal region.

The fi rst evidence for duplicated sequences came from the identifi ca- tion of a gene family (D15F37/HERC2), which was found to have mul- tiple expressed copies within the breakpoint cluster regions in proximal 15q.

Using combinations of molecular and cytogenetic methods,

it could be demonstrated that all duplicated copies of this gene family and additional expressed and nonexpressed sequences are part of large duplicated sequence stretches of 200–400 kb in size.

Balanced translocations involving chromosome 15 are extremely rare in PWS. So far, fi ve paternally derived de novo translocations have been reported, all of which disrupt the SNURF-SNRPN gene in either of two locations (Figure 3.1). In two patients with typical PWS, the translocation breakpoint disrupts SNURF.

In three patients with some features of PWS, the translocation breakpoints clustered in a region distal to SNRPN.

Maternal Uniparental Disomy

To fi nd out more about the molecular defect in PWS patients without

del(15)(q11q13), Nicholls, Butler and colleagues in 1989 studied DNA

samples from such families with the help of chromosome 15 markers.

They identifi ed two probands that lacked a paternal chromosome 15 and found that they had two different maternal chromosome 15 s [uni- parental heterodisomy 15, upd(15)mat]. This fi nding established that the PWS chromosomal region is subject to genomic imprinting and that PWS is caused by the absence of a paternal contribution of genes on chromosome 15. As the paternally expressed genes are silent on the maternal chromosome, the loss of a paternal chromosome cannot be com- pensated by a second maternal chromosome 15. Subsequent studies revealed that upd(15)mat is the second most frequent fi nding in PWS and accounts for approximately 25% of all cases.

Maternal uniparental disomy arises in most cases from the postzy- gotic correction of a meiotic error. During meiosis, the diploid set of chromosomes (n = 46) is reduced to a haploid set (n = 23). Nondisjunc- tion of the homologous chromosome 15 s during female meiosis I or nondisjunction of the two sister chromatids during meiosis II results in an oocyte with two chromosome 15 s or no chromosome 15. Fertiliza- tion of an oocyte with two chromosome 15 s by a normal sperm with one chromosome 15 leads to a zygote trisomic for chromosome 15. This condition is not compatible with normal development but can be rescued by loss of one chromosome 15. In two thirds of cases, one of the two maternal chromosome 15 s will be lost from the trisomic cell.

This results in a normal set of chromosomes. If, however, the paternal chromosome is lost, the cell is left with two maternal chromosomes [upd(15)mat]. As judged by the state of DNA markers close to the centromere, most cases result from a meiosis I error. In these cases, the marker closest to the centromere is heterozygous. If the patient is homozygous at this marker, although the mother is heterozygous, a meiosis II error is likely. As a consequence of recombination events prior to the segregation of the chromosomes, heterodisomic and isodi- somic segments alternate along the chromosome. As in other non- disjunction cases, the risk of uniparental disomy (UPD) increases with maternal age.

The recurrence risk is very low if the parents have normal chromosome 15 s.

Imprinting Defects

A few patients with PWS (about 1%) have apparently normal chromo- some 15 s of biparental inheritance, but the paternal chromosome carries a maternal imprint. This leads to a complete loss of the pater- nally expressed genes in 15q11–q13. Thus, the functional consequence of the incorrect imprint is identical to that of maternal uniparental disomy.

In approximately 15% of these patients with PWS and an imprinting defect, the incorrect imprint is the result of a microdeletion affecting the imprinting center (IC).

The IC overlaps with the SNURF- SNRPN gene (Figure 3.1) and regulates in cis DNA methylation, gene expression, and chromatin structure of the whole imprinted domain.

The IC appears to consist of two elements. One element is defi ned by

Table 3.1. Estimated Frequency and Recurrence Risk for Specifi c Genetic Subtypes in Prader-Willi Syndrome

Deletion

15q11–q13 Uniparental Imprinting Balanced (de novo) disomy 15 defect translocations

Frequency 70% 25% ~1% <0.1%

Recurrence risk <1% <1% 0–50% Unknown

IC deletions in patients with AS and an imprinting defect. The shortest region of overlap (AS-SRO) is 880 bp and maps 35 kb centromeric to the SNURF-SNRPN.

The IC element affected by these deletions is neces- sary for establishing the maternal imprint. It probably interacts with the second element, which is defi ned by IC deletions in patients with PWS and an imprinting defect. The shortest region of overlap (PWS- SRO) is 4.3 kb in size and spans exon 1 of SNURF-SNRPN.

The IC element affected by these deletions is required for the maintenance of the paternal imprint during early embryogenesis.

Most of the IC deletions are familial mutations. A deletion of the PWS-SRO can be transmitted silently through the female germ line, but leads to an incorrect maternal imprint on the paternal chromo- some when inherited from a male. Familial IC deletions are associated with a 50% recurrence risk (see Table 3.1). In the case of a de novo deletion, the recurrence risk is not increased when it occurred after fertilization, but it can be up to 50% when the father has a germ line mosaicism.

The majority of patients with PWS and an imprinting defect (85%) have no IC deletion or point mutation of the PWS-SRO.

This indi- cates that the imprinting defect occurred spontaneously in the absence of a DNA sequence change. Interestingly, in all informative cases the chromosome carrying the incorrect imprint was inherited from the paternal grandmother.

These data suggest that the imprinting defect results from a failure to erase the maternal imprint in the father’s germ line. In contrast to patients with an IC deletion, some of these patients share the same paternal chromosome with a healthy sibling. This indicates that the recurrence risk for another child with the disorder is very low.

Genes in the 15q11–q13 Region

The proof for genomic imprinting of 15q11–q13 has come from the

identifi cation of genes that are expressed from the paternal or maternal

chromosome only (Figure 3.1). Within the 2 Mb imprinted domain, four

paternally expressed genes encoding a protein have been identifi ed,

MKRN3, MAGEL2, NDN, and the SNURF-SNRPN. Paternal-only

expression of these genes is regulated by parent-of-origin-specifi c DNA methylation of the promoter regions of each gene. Whereas the active paternal allele is unmethylated, the inactive maternal allele is methyl- ated. Parent-of-origin-specifi c DNA methylation can be used to confi rm the clinical diagnosis of PWS patients with a deletion of 15q11–q13, uniparental disomy, or an imprinting defect. Recently, several pater- nally expressed small nucleolar (sno) RNA genes have been identifi ed.

Telomeric to the paternally expressed domain are two maternally expressed genes, UBE3A

and ATP10C.

MAKORIN3 (MKRN3, formerly ZNF127) is a ubiquitously expressed intronless gene that defi nes the most telomeric border of the imprinted domain of human 15q11–q13.

It encodes a putative RING zinc fi nger transcription factor, which belongs to the MAKORIN gene family and may function as a ribonucleoprotein. Just telomeric to MKRN3 are two other intronless genes, MAGEL2 and NECDIN (NDN). Both genes encode proteins that are part of the melanoma-associated antigen (MAGE) protein family.

MAGEL2 is expressed only in the brain and placenta.

In the mouse, Necdin (Ndn) is expressed predominantly in postmi- totic neurons with the highest expression in the hypothalamus and other brain regions at late embryonic and early postnatal stages.

However, the human NDN gene was found to be expressed in all tissues studied, with highest expression in the brain and placenta. It is upregulated during neuronal differentiation, and in vitro experiments have shown that over-expression of this gene leads to suppression of cell proliferation. Data obtained from different mouse models suggest that this gene may contribute to respiratory problems observed in patients with PWS (see below).

The most complex gene in 15q11–q13 is SNURF-SNRPN. The origi- nal gene was found to consist of 10 exons, which encode two different proteins.

Exons 1 to 3 encode SNURF, a small polypeptide of unknown function, while exons 4 to 10 encode SmN, a spliceosomal protein involved in mRNA splicing in the brain. Exon 1 and the pro- moter region overlap with the IC. In the past few years, many more 5¢ and 3¢ exons of SNURF-SNRPN have been identifi ed. These exons have two peculiar features: they do not have any protein coding poten- tial, and they occur in many different splice forms of the primary transcript. Alternative transcripts containing novel 5¢ exons were described by Dittrich et al.

and characterized in detail by Färber et al.

These transcripts start at two sites that share a high degree of sequence similarity and span the AS-SRO. Additional 3¢ exons were described by Buiting et al.

and Runte et al.

This analysis also showed that the IPW exons, which were previously thought to represent an independent gene,

are part of the SNURF-SNRPN transcription unit.

Some of these splice variants are found predominately in the brain and

span the UBE3A gene in an antisense orientation. In contrast to the

paternally expressed genes in 15q11–q13, maternal-only expression of

UBE3A in brain is not regulated by DNA methylation. It is tempting

to speculate that the antisense transcript silences the paternal allele of

UBE3A.

Interestingly, the IC-SNURF-SNRPN transcript also serves as a host for several snoRNAs, which are encoded within introns of this complex transcription unit. They are processed from the primary transcript, and as this is made from the paternal chromosome only, the snoRNAs show the same imprinted expression pattern. The genes are present as single copy genes (HBII-13, HBII-437, HBII438A and HBII-438B) or as multi-gene clusters (HBII-85 with 27 gene copies and HBII-52 with 47 gene copies).

In contrast to other snoRNAs, which are usually involved in the modifi cation of ribosomal RNAs, these snoRNAs do not have a region complementary to ribosomal RNA and might be involved in the modifi cation of mRNAs. In HBII-52, 18 nucleotides are complementary to the serotonin receptor 2C mRNA.

It is possible that these snoRNAs are involved in the editing and/or alternative splicing of this mRNA.

In two unrelated families, a small deletion spanning UBE3A and the HBII-52 gene cluster has been identifi ed. Whereas maternal transmis- sion of the deletion leads to AS, paternal transmission is not associated with an obvious clinical phenotype. This excludes the HBII-52 snoRNAs from a role in PWS. The HBII-85 gene cluster is distal to three balanced translocation breakpoints in patients with some features of PWS. As HBII-85 is not expressed in these patients,

these snoRNAs may play a role in PWS.

In addition to the imprinted genes, several nonimprinted genes have been identifi ed in the chromosomal region affected by the common large deletion (Figure 3.1). These genes may modify the PWS pheno- type, and some are responsible for other genetic disorders. One is the oculocutaneous albinism type II (OCA2) gene. Hypopigmentation is a frequent fi nding in PWS and AS patients with a common large dele- tion.

This nonimprinted phenotype is associated with the deletion of one OCA2 gene copy and may be caused by a gene dosage effect.

However, Bittel et al.

did not obtain any evidence, by expression profi ling, of reduced OCA2 mRNA levels in lymphoblastoid cell lines from PWS and AS deletion patients. They did observe several tran- scripts from within the PWS/AS region, e.g., the GABA receptor subunit genes GABRA5 and GABRB3—that had less than half the control level of expression in the deletion cell lines. These genes are probably expressed at higher levels from the paternal allele than the maternal allele. A maternal bias of expression was seen for four genes including UBE3A and ATP10C. Different expression levels of some of these genes in UPD versus deletion cells may underlie the phenotypic differences seen in PWS patients with different chromosomal defects.

Patients with a class or type I deletion, but not patients with a type

II deletion, are hemizygous for four nonimprinted genes located

between the deletion breakpoint cluster regions BP1 and BP2. These

are the genes NIPA1, NIPA2, CYFIP1, and GCP5.

Heterozygous muta-

tions in NIPA1 lead to autosomal dominant spastic paraplegia.

Since

spastic paraplegia has never been observed in patients with PWS or AS

and a type I deletion, it is likely that a mutant protein and not reduced

gene dosage leads to the disorder. Recently, clinical differences have

been reported by Butler et al.

in PWS subjects with type I deletions

versus type II deletions. Type I deletion subjects have more behavioral problems than type II deletion PWS subjects.

Downstream Genes from the 15q11–q13 Region

It is likely that the loss of function of the paternally expressed genes in 15q11–q13 directly or indirectly affects the activity of other genes. The identifi cation of downstream genes may help to understand the patho- genesis of PWS. To date, only one study has addressed this question.

Using high density microarrays, Horsthemke et al.

compared the gene expression profi les of normal and upd(15) fi broblasts isolated from a mosaic patient. By comparing cell lines from the same individual, Horsthemke et al.

circumvented the problem of interindividual vari- ation in gene expression. However, there are also certain drawbacks associated with the study: cloned fi broblasts can show considerable interstrain variation, and the major symptoms in PWS are related to brain and not fi broblast dysfunction. The overall gene expression pro- fi les were highly similar, indicating that the chromosome 15 status did not have a major effect on the global gene expression pattern in fi brob- lasts. Among the genes showing reduced expression in upd(15)mat cells was SCG2, which encodes for secretogranin II. This protein is mainly found in the core of catecholamine-storage vesicles within cells of the neuroendocrine system. It is a precursor of secretoneurin, which induces dopamine release.

SCG2 is an interesting candidate gene, because dopamine is known to regulate food intake by modulating food reward via the meso-limbic circuitry of the brain.

Recent imaging studies in humans

have linked dopamine with eating behavior and obesity, and Wang et al.

have suggested that obese individuals may perpetuate pathological eating as a means to compensate for a decreased reward. To substantiate the notion that a defect in dopamine- modulated food reward circuits contributes to the development of hyperphagia in PWS, it will be necessary to replicate these fi ndings in other patients and in mouse models. In addition, Bittel et al.

reported altered expression of genes/transcripts distal to the 15q11–q13 region using microarray technology in PWS and AS deletion subjects, which may refl ect the different chromatin patterns in these subjects compared with normal.

Mouse Models of Prader-Willi Syndrome

Human chromosome region 15q11–q13 is evolutionally related to mouse chromosome region 7C. The conserved synteny of the “PWS genes” makes it possible to study the effects of uniparental disomy and chromosomal deletions, as well as knock-outs for individual genes and regulatory elements in mice. These studies may help to unravel the pathogenesis of PWS.

The fi rst mouse model for maternal uniparental disomy was described

by Cattanach and colleagues in 1992.

Mice with a maternal duplica-

tion of 7C are smaller compared with their wild type littermates and die 2 to 8 days after birth. The early postnatal lethality is possibly associated with a reduced suckling activity. This effect is therefore consistent with the feeding problems and failure-to-thrive in newborns with PWS.

Nicholls and colleagues in 1999 reported a transgene mouse model with a chromosome deletion of 7C.

The size of the deletion is similar to the deletions in PWS patients. Mice inheriting the deletion from their father show failure-to-thrive and growth retardation and die within the fi rst week of life.

Several knock-outs of individual genes have been described also.

Jong et al.

demonstrated that a disruption of Mkrn3 had no pheno- typic effect in the mouse. The resulting mouse was viable and fertile, suggesting that Mkrn3 does not have a signifi cant role in PWS.

The Ndn gene was studied in three different mouse models. In the fi rst study,

no obvious phenotype was seen. However, in two other studies, Gerard et al.

and Muscatelli et al.

found that in certain mouse strains the paternal transmission of a null allele strain leads to postnatal lethality with variable penetrance. The lethality of the mutant mice arose from a respiratory defect, suggesting that absence of NDN expression may contribute to the observed respiratory abnormalities in individuals with PWS through a suppression of central respiratory drive.

Surviving mice in all three studies had no overt phenotype and were non-obese and fertile.

Furthermore, Muscatelli et al.

found behavioral and hypothalamic alterations in Ndn-defi cient mice: Ndn mutant mice displayed increased skin scraping in an open fi eld test and improved spatial learning and memory in the Morris water maze, possibly mimicking the skin picking and improved spatial memory typical of PWS. These fi ndings suggest that the NECDIN protein may be responsible for at least a subset of the multiple clinical features present in PWS.

Brannan and her colleagues showed that a disruption of the Snrpn gene is without any obvious phenotypic effect.

The same is true for a Snurf deletion, as demonstrated by Tsai et al.,

but White et al.

sug- gested that the Snrpn-defi cient mice may have a defi ciency of dopamine in the striatum.

To study the IC, which overlaps with Snrpn exon 1, three different deletions were engineered. Bressler et al.

found that a 0.9 kb deletion of the exon 1 region of Snurf-Snrpn had no effect, whereas a paternally transmitted 4.8 kb deletion led to postnatal lethality in about 50% of mutant mice. Surviving mutant mice were viable and healthy.

The paternal transmission of a 42 kb deletion (exons 1–6 of Snurf- Snrpn plus 23 kb upstream sequence) resulted in 100% postnatal lethality. The mutant mice were smaller than their wild type litter- mates, exhibited hypotonia and died within the fi rst days of life. Again, strain-specifi c differences were observed.

By breeding chimeric males with females of another strain, some viable offspring were obtained.

Surviving mice were smaller than normal, but fertile and not obese.

Expression studies of the paternally expressed genes at birth, 1 week,

2 weeks, and 3 weeks of age revealed a leaky expression of all these

genes. Even more surprising was the fi nding that, based on studies of the Snrpn locus, the leaky expression was derived from the maternal allele. From this it can be concluded that certain mouse strains do not silence imprinted genes as completely as other strains. Furthermore, it appears that only low levels of expression of PWS candidate genes are required to overcome most of the typical features of PWS.

Two mouse models with a deletion distal to the Snurf-Snrpn gene have been reported. One deletion spans from Snurf-Snrpn exon 2 to Ube3a.

Mice with a paternally inherited deletion showed hypotonia, growth retardation, and 80% postnatal lethality. All surviving animals were fertile and non-obese. No obvious phenotype was found in mice harboring a paternally derived deletion spanning from the Ipw exons to Ube3a.

This suggests that the critical region for many PWS features maps between Snrpn and the Ipw exons. This interval does not contain any protein-coding genes but only the snoRNA genes MBII-85, MBII-13 and MBII-437. Therefore, these snoRNAs are good candidates for hypo- tonia, failure-to-thrive, and growth retardation.

A common feature in the mouse models that showed a phenotypic effect is postnatal lethality, and apart from the Ndn-defi cient mice, all exhibit growth retardation. Only the Ndn-mutant mice showed hypoth- alamic alterations and features mimicking respiratory problems in PWS babies. Hypotonia was reported for both an IC-deletion mouse and a deletion including snoRNA genes. In none of the mouse models was obesity and/or hypogonadism found. However, the UPD and large deletion mice do not survive the fi rst week of life, and therefore these animals cannot be studied at later developmental stages.

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