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An imprinted gene p57KIP2 is mutated in Beckwith- Wiedemann syndrome
Izuho Hatada1*, Hirofumi Ohashi2, Yoshimitsu Fukushima3, Yasuhiko Kaneko4, Masahiro Inoue5, Yosuke Komoto5, Akira Okada5, Sachiko Ohishi1, Akira Nabetani1, Hiroko Morisaki1, Masahiro Nakayama6,
Norio Niikawa7 & Tsunehiro Mukai1
p57KIP2 is a potent tight-binding inhibitor of sever- al G1 cyclin/Cdk complexes, and is a negative reg- ulator of cell proliferation1,2. The gene encoding p57KIP2 is located at 11p15.5 (ref. 2), a region impli- cated in both sporadic cancers and Beckwith- Wiedemann syndrome, a cancer-predisposing syndrome, making it a tumour-suppressor candi- date. Several types of childhood tumours including Wilms’ tumour, adrenocortical carcinoma and rhab- domyosarcoma exhibit a specific loss of maternal 11p15 alleles, suggesting that genomic imprinting3-8 is involved9-12. Genetic analysis of the Beckwith- Wiedemann syndrome indicated maternal carriers, as well as suggesting a role of genomic imprint- ing13. Previously, we and others demonstrated that p57KIP2 is imprinted and that only the maternal allele is expressed in both mice and humans14-16. Here we describe p57KIP2 mutations in patients with Beckwith-Wiedemann syndrome. Among nine patients we examined, two were heterozygous for different mutations in this gene - a missense muta- tion in the Cdk inhibitory domain resulting in loss of most of the protein, and a frameshift resulting in disruption of the QT domain. The missense muta- tion was transmitted from the patient’s carrier moth- er, indicating that the expressed maternal allele was mutant and that the repressed paternal allele was normal. Consequently, little or no active p57KIP2 should exist and this probably causes the over- growth in this BWS patient.
Į National Cardiovascular Center Research Institute, 5-7-1, Fujishiro-dai, Suita, Osaka 565, Japan 2Saitama Children’s Medical Center, 2100 Magome, Iwatsuki, Saitama 339, Japan 3Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390, Japan 4Saitama Cancer Center, 818 Komuro, Ina, Saitama 362, Japan 5 Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan 6Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodou-cyo, Izumi, Osaka 565, Japan 7Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki 852, Japan
Correspondence should be addressed to I.H. e-mail: hatada@ ri.ncvc.go.jp
Progression through the cell cycle is catalysed by cyclin- dependent kinases (CDKs) and is negatively controlled by CDK inhibitors (CDKIs); p57KIP2 is related to p21 CIPI and p27KIP2, and is a potent tight-binding inhibitor of several G1 cyclin/CDK complexes1,2. Overexpression of p57KIP2 arrests cells in G1. The gene encoding human p57KIP2 is located on chromosome 11p15.5 (ref. 12), a region impli- cated in both Beckwith-Wiedemann syndrome (BWS) and sporadic cancers. BWS is characterized by numerous growth abnormalities, including macroglossia, gigantism, visceromegely, exomphalos and an increased risk of child- hood tumours, including Wilms’ tumour, adrenocortical carcinoma, rhabdomyosarcoma and hepatocellular carci- noma17. Although most cases of BWS are karyotypically normal and sporadic, there are patients with chromosome 11 duplications18,19 or translocations20 and a few families with autosomal dominant transmission21. Evidence that the gene for BWS is imprinted comes from the increased maternal transmission pattern seen in the autosomal dom- inant-type pedigrees22,23 and especially from the findings of paternal uniparental disomy (UPD) reported for a sub-
group of patients24. The region most commonly involved in uniparental disomy includes the gene for p57KIP2 on 11p15.5 (ref. 24). The gene for BWS has also been local- ized to the 11p15.5 region by linkage analysis of autoso- mal dominant pedigrees25,26. We have demonstrated that p57KIP2 is imprinted in both mice and humans14-16. Interestingly, experiments using subchromosomal trans- ferable fragments from 11p15 have shown that a tumour suppressor gene maps in the vicinity of D11S724 and D11S719, excluding H19 (ref. 27), suggesting that p57KIP2 might be an imprinted tumour suppressor in this region. Among the eight breakpoints of BWS patients examined, five were mapped close to cC15-19 and q1 (ref. 28). If the tumour suppressor and BWS gene are identical, these findings suggest that the gene is located in the vicinity of D11S724 and q1. p57KIP2 is located very close to D11S679, between D11S724 and q1 (ref. 15).
We analysed DNA samples from nine unrelated Japanese BWS patients for mutations by direct DNA sequencing of PCR-amplified products. The human gene encodes a 316 amino-acid protein consisting of three structurally distinct domains1,2: an N-terminal Cdk-inhibitory domain with significant similarity to p21 CIPI (refs 29, 30) and p27KIPI (ref. 31); a region containing proline-alanine repeats (PAPA repeats) and a C-terminal domain conserved with p27KIPI (QT domain). The entire coding region of p57KIP2, includ- ing intron/exon boundaries, was analysed by direct DNA sequencing using five PCR primer pairs. Mutations were detected in two patients (patients 6 and 8). Patient 6 was a 7-year-old boy diagnosed as having BWS based on the fol- lowing features: increased birth weight, omphalocele, macrogrossia, intractable neonatal hypoglycaemia, facial nevus flammeus, and ear lobe grooves. Both parents, his grandparents and his sister are healthy. In this patient, PCR amplification and direct sequencing analysis revealed a het- erozygous C to T transition at nucleotide 399 changing a glutamine (CAG) to a termination (TAG) codon in codon 47 (Fig. la). This would result in a severely truncated polypeptide of 46 residues with disruption of the Cdk inhibitory domain and loss of the QT domain and the PAPA repeats (Fig. 2). As this mutation disrupts a PstI site, the presence of the mutation was confirmed by restriction enzyme analysis. Digestion of the PCR amplified-DNA of the patient with PstI gave a novel 219-bp fragment, in addi- tion to three other fragments (126, 93 and 60 bp) which could also be detected in normal controls (Fig. 3a).
Patient 8 was a 3-month-old girl. She was diagnosed as having BWS based on the following features: gigantism, omphalocele, macrogrossia, neonatal hypoglycaemia and ear lobe grooves and pits. Both parents and grandparents are healthy. Direct sequencing analysis and sequencing of each cloned allele revealed a heterozygous T to AG trans- version/addition at nucleotide 1086 that modified the nine amino acids downstream and resulted in a premature translation termination (Fig. 1b). This would result in a truncated polypeptide of 284 residues with disruption of the QT domain (Fig. 2). As this mutation disrupts a MboII site, the presence of the mutation was confirmed by restriction enzyme analysis (data not shown).
Familial cases of BWS are inherited in an autosomal dominant mode but exclusively through the mother13. Therefore, we examined whether the mutation in patient 6 was derived from his father or mother. We digested the PCR-amplified DNA of the parents with PstI. As expected, the mother also had the 219-bp fragment (mutant allele)
npg
a
CGCGAGCTG CAGGCCCGC
CGCGAGCTGÇAGGCCCGC
TANA SAME
Control
Patient 6
RELQAR
Patient 6
Normal CGC GAG CTG CAG GCC CGC Mutant CGC GAG CTG TAG GCC CGC
REL *
b
TTCT TCG CCAAG CO CAAG AG ATCAOCOCCTO AG
TTCAGTCOCCAAGCOCAAG AGATCAGCOCCTO A
Control
Mutant (Patient 8)
FFAKRKRSAPE Normal TTC TTC GCC AAG CGC AAG AGA TCA GCG CCT GAG Mutant TTC AGT CGC CAA GCG CAA GAG ATC AGC GCC TGA
Patient 8
FSRQAQEISA *
whereas the father only had a normal allele. The mother was heterozygous for the mutant allele as was the patient. The mother is a designated carrier because she has a normal phenotype. Her mutant allele is speculated to have been inherited from her father; its expression would be repressed and the normal allele expressed - because p57KIP2 is expressed predominantly from the maternal allele14-16. In contrast, in the patient the expressed maternal allele was mutant and the repressed paternal allele was normal (Fig. 3b). Consequently, little or no active p57KIP2 protein should exist and this seems to cause the overgrowth in this BWS patient. (Recently, the p27KIPI gene related to p57KIP2 was targeted in mice, resulting in overgrowth of the body32-34, consistent with that observed in our patients.)
The seven BWS patients who have no mutation could be due to the reduced expression of p57KIP2 by paternal uniparental disomy, loss of imprinting or maternal translocation. Among the two patients in whom we could examine gene expression, patient 7 showed reduced
Cdk inhibitory domain PAPA repeats
QT domain
Normal
Patient 6
Mutant
Patient 8
Fig. 1 Identification of p57KIP2 mutations. a, Sequence analysis of DNA from the control and patient 6. A heterozygous C to T tran- sition at nucleotide 399, changing a glutamine (CAG) to a termi- nation (TAG) codon in codon 47 (the Cdk inhibitory domain) is observed. b, Sequence analysis of cloned DNA from control and patient 8. Normal allele from patient 8 is the same as the control (not shown) and only the mutant allele is shown. A transver- sion/addition (T to AG) at nucleotide 1086 that modifies the nine downstream amino acids is observed. This results in a premature translation termination in the QT domain.
expression in her adrenal gland (data not shown). Anoth- er possibility is involvement of other loci. There are three other known BWS balanced translocations that map sev- eral megabases from this region28. IGF2 could also be involved because we could not explain the aetiology of paternal duplications by p57KIP2 as, in this class, a mater- nal allele is present and presumably functional.
p57KIP2 also is a tumour-suppressor candidate for Wilms’ tumour. A tumour-suppressor gene has been mapped to the vicinity of D11S724 and D11S719 includ- ing p57KIP2 through experiments using subchromoso- mal transferable fragments from 11p15 (ref. 27). Overexpression of p57KIP2 arrests cells in G1 (ref. 2) and we reported the reduced expression of p57KIP2 in Wilms’ tumours15. However, involvement of other genes in 11p15.5 is possible because the association of BWS with Wilms’ tumour is rather mild. Therefore, it is important to find mutations in Wilms’ tumours and to examine whether introduction of p57KIP2 to Wilms’ tumour cells can suppress the tumour phenotype.
Our results represent the first direct demonstration that an imprinted gene causes a human disease. It also illustrates a new mechanism for producing a phenotype with dominant transmission with little or no gene prod- uct. One allele with an inactive product is expressed and the other allele is repressed by genomic imprinting.
Methods
PCR amplification and direct DNA sequencing. The p57KIP2 gene was examined for mutations by direct sequencing of 5 PCR-amplified fragments. The primers used were: fragment 1, 5’-CGTTCCACAGGCCAAGTGCG-3’ and 5’-GCTGGTGCG- CACTAGTACTG-3’; fragment 2, 5’-CGTCCCTCCGCAGCA- CATCC-3’ and 5’-CCTGCACCGTCTCGCGGTAG-3’; frag- ment 3, 5’-TGGACCGAAGTGGACAGCGA-3’ and 5’-GG- GGCCAGGACCGCGACC-3’; fragment 4, 5’-CGGAATTCCG- GAGCAGCTGCCTAGTGTC-3’ and 5’-CTTTAATGCCAC- GGGAGGAGG-3’; fragment 5, 5’-CGGCGACGTAAACAA- AGCTG-3’ and 5’-GGTTGCTGCTACATGAACGG-3’.
The reaction mixtures used were: fragment 1, 10 mM Tris- HCI, pH 8.3, 50 mM KCI, 2.5 mM MgCl2, 0.2 mM dATP, dGTP, dCTP and dTTP, 5% dimethyl sulphoxide and 2.5 U Taq poly- merase in a final volume of 50 pl; fragments 2, 3, 5, 10 mM Tris- HCI, pH 8.3, 50 mM KCI, 1.5 mM MgCl2, 0.2 mM dATP, dGTP, dCTP and dTTP, 5% dimethyl sulphoxide and 2.5 U Taq Polymerase in a final volume of 50 ul; fragment 4, 1x LA PCR Buffer II (Takara), 0.2 mM dATP, 7-deaza dGTP, dCTP and dTTP, 5% dimethyl sulphoxide and 2.5 U Taq polymerase in a final volume of 50 ul. Temperature conditions used were: for fragments 1, 2, and 5, an initial step at 95 ℃ for 4 min, 40 cycles of 95 ℃ (1 min), 65 ℃ (1 min) and 72 ℃ (1 min), followed by a 10-min extension at 72 °℃; for fragment 3, an initial step at 95 ℃ for 4 min, 40 cycles of 95 ℃ (1 min), 70 ℃ (1 min) and 72 ℃ (1 min), followed by a 10-min extension at 72 ℃; for frag- ment 4, an initial step at 95 ℃ for 4 min, 40 cycles of 95 ℃ (1 min), 65 ℃ (1 min) and 72 ℃ (1.5 min), followed by a 10-min extension at 72 ℃. PCR products were fractionated by elec- trophoresis on a 2% Seakem GTG agarose gel. The amplified bands were excised from the gel and purified using a QIAEX II
npg
a
b
Pstl
Pstl
Normal
126 bp
93 bp
60 bp
E
八
-
E
Pstl
Mutant
219 bp
60 bp
3
9
?
Normal
Normal
Patient
Father
Mother
Father (Normal)
Mother (Carrier)
219 bp
Repressed p57KIP2
=
Expressed p57KIP2
126 bp
Mutated p57KIP2
9
93 bp
60 bp
Patient
Gel Extraction Kit (QIAGEN). Sequencing was performed using a ABI PRISM dye terminator cycle sequencing kit (ABI).
Acknowledgements
We thank S. Miyabara for useful advice and K. Tohyama for technical assistance. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of
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