ELSEVIER
Li-Fraumeni syndrome in a Malaysian kindred
Hany Ariffinª,*, Ghyslaine Martel-Plancheb, Siti Sarah Dauda, Kamariah Ibrahima, Pierre Hainautb
aPediatric Hematology-Oncology Unit, Department of Pediatrics, University of Malaya Medical Center, 50603 Kuala Lumpur, Malaysia bGroup of Molecular Carcinogenesis and Biomarkers, International Agency for Research on Cancer (IARC), 150 Cours Albert Thomas, 69372 Lyon, France Received 2 May 2008; received in revised form 21 May 2008; accepted 11 June 2008
Abstract
We report on a Malaysian kindred with Li-Fraumeni syndrome. The proband was an 8-year-old girl who presented with embryonal rhabdomyosarcoma of the trunk at the age of 8 months and de- veloped a brain recurrence at the age of 7 years, which was 5 years after remission. A younger sister later developed adrenocortical carcinoma at the age of 6 months. Their mother and maternal grand- mother were diagnosed with breast cancer at the ages of 26 and 38 years, respectively. TP53 mu- tation detection in this family revealed a duplication of a GGCGTG motif starting at nucleotide 17579 in exon 10, resulting in an in-frame insertion of two amino acids between residues 334 and 336 in the tetramerization domain of the p53 protein. This mutation was found in the proband and her affected sister as well as her mother. In addition, the mutation was detected in two other siblings (a brother aged 3 years and a sister aged 18 months) who have not yet developed any ma- lignancy. Sequencing of TP53 in the father and two other asymptomatic siblings revealed wild-type TP53. To our knowledge, this is a first report of a Li-Fraumeni syndrome family in Southeast Asia. @ 2008 Elsevier Inc. All rights reserved.
1. Introduction
The Li-Fraumeni syndrome (LFS) is a rare autosomal familial cancer syndrome characterized by an increase inci- dence of malignancies in family members aged younger than 45 years. The commonest cancers associated with LFS are bone and soft tissue sarcomas, breast carcinoma, brain tumors and adrenocortical carcinoma. Other cancers include leukemia, gastric, colorectal, and pancreatic cancer, choroid plexus papilloma, and Wilms tumors.
The criteria originally described by Li et al. [1] in 1969 were as follows: a proband with a sarcoma before the age of 45 years, a first-degree relative of the proband with any cancer before age 45, and another first- or second-degree rel- ative in the same lineage with any cancer before age 45 or sar- coma at any age. Later, Chompret et al. [2] revised these criteria to include any child with adrenocortical carcinoma.
In 1990, the association between LFS and germline mu- tations of the TP53 tumor suppressor gene was demon- strated [3,4]. Since this original report, mutations scattered throughout the coding sequence of the gene have been
consistently reported in ~70% of LFS families, as well as in some 20-50% of families with familial cancer syn- dromes that present LFS traits but not fully matching Chom- pret criteria (often referred to as Li-Fraumeni-like) [5,6]. Most of these mutations are missense, resulting in amino acid substitutions within the DNA binding domain of the p53 protein, crippling its capacity to bind to DNA and to act as a tumor suppressive transcription factor. Recently, it was found that large deletions encompassing the whole gene or large regions of its coding and non-coding sequences, may also occur in some families [5]. These alterations are not easily detected by DNA sequencing. All known germ- line TP53 alterations associated with inheritance of cancer are compiled in the IARC TP53 database [7]. All LFS fam- ilies described to date originate from Western Europe, North America and, more recently, South America. So far, no case of LFS has been reported from Southeast Asia. We report an affected kindred from the Malay ethnic group.
2. Materials and methods
2.1. Case report
The proband, an 8-year-old girl, first presented at the age of 8 months with a truncal mass that was proven histologically
* Corresponding author. Tel .: +60-3-7949-2065; fax: +60-3-7955- 6114.
E-mail address: hany@um.edu.my (H. Ariffin).
to be embryonal rhabdomyosarcoma. She underwent pri- mary surgery, followed by chemotherapy according to the SIOP MMT89 protocol. After four courses, her parents re- fused further therapy. She returned 17 months later with a local recurrence. She received further chemotherapy, fol- lowed by surgical resection of the tumor and irradiation of the tumor bed.
Two years later, her 6-month-old sister was diagnosed to have left adrenocortical carcinoma, after an incidental find- ing of an abdominal mass. She underwent surgical resection and chemotherapy consisting of oral mitotane and six courses of etoposide and cisplatinum.
In the same year as the presentation of the younger sib- ling, their mother (then aged 26 years) was diagnosed to have carcinoma of the right breast, for which she underwent a mastectomy. A year later, she developed carcinoma in the left breast and underwent mastectomy and chemotherapy. As of writing, both mother and younger daughter had re- mained in continuous clinical remission through four years after completion of all therapy.
Seven years after her first presentation, the proband returned with right-sided focal seizures. Radioimaging revealed a left temporoparietal mass, which was surgically debulked. This was histologically proven to be a recurrence of rhabdomyosarcoma. She received six courses of topote- can and cyclophosphamide as salvage therapy; however, despite initial response, the tumor progressed in size and the child died of disease 10 months after cessation of therapy.
2.2. Molecular genetic testing
Because the proband fulfilled the criteria of LFS, we wanted to determine whether this family carried a TP53 mutation. Briefly, after counseling and informed consent, genomic DNA was extracted from peripheral blood leuko- cytes from both parents and the affected children. At the
parents’ request, DNA was also obtained from their four other unaffected children, aged between 1 and 12 years (Fig. 1). Exons 2 to 11 of the TP53 gene and their flanking splice site junctions were amplified using primers and proto- cols available from the International Agency for Research on Cancer (IARC) website [8], followed by bidirectional auto- mated DNA direct sequencing. Mutation findings were con- firmed by sequencing of a second, independent polymerase chain reaction product from the proband’s genomic DNA.
3. Results
The TP53 sequence is GGG CGT GAG, so the inserted sequence should read G(GG CGT G)GG CGT GAG.
We found a previously unreported duplication of a 6-bp motif (GGCGTG) starting at the second base of codon 334 (nucleotide 17579 in exon 10) (Fig. 2). This duplication re- sulted in an in-frame insertion of two amino acid residues, repeating residues 334 (Gly) and 335 (Arg). This alteration was found in the proband, in her affected sister, and in her mother. In addition, the mutation was detected in two other siblings (a brother aged 3 years and a sister aged 18 months) who have yet to manifest any malignancy. Sequencing of TP53 from the father and two other siblings revealed wild- type sequences.
4. Discussion
We report a novel, unconventional mutation in a Malay- sian family with LFS. This mutation consists in a 6-bp du- plication that induces a repetition of residues 334 (Gly) and 335 (Arg), modifying the structure of a large a-helix in the oligomerization domain of p53, which is involved for the formation of p53 dimers and tetramers. The a-helixes of two p53 monomers align antiparallel to each other, and this arrangement is stabilized by the donation of a hydrogen
I
1
2
3
4
Breast cancer (38 yr)
II
1
2
3
4
5
6
7
8
9
10
11
12
13
30 yr
Breast cancer (26 yr)
III
1
2
4
5
6
Embryonal RMS (8 mo)
3
Adrenocortical carcinoma (6 mo)
A
AT CC GT G G GC GT GAGC GCTTC
50
60
B
G
T G
AT CC GT G G G C GT GAG CGCNN
50
60
C
bond from Arg337 on one a-helix to Asn352 on the other helix. We postulate that the mutation perturbs the correct folding of the a-helix, preventing Arg337 to fall in the proper place and orientation to donate a hydrogen to Asn352 on the other helix, thus precluding the assembly of p53 into oligomers with high DNA binding and transac- tivational capacity.
There is evidence that mutation that hampers hydrogen donation by Arg337 may cause LFS or Li-Fraumeni-like syndromes. In southern Brazil, several kindreds have been described as carriers of the same mutation at codon 337, likely due to a founder effect [9]. This mutation induces the replacement of Arg at position 337 by His. While the latter residue can still donate a hydrogen to Asn352, it can do so only at pH conditions in the lower physiological range (≤7-7.5), whereas at pH 8 the histidine is deproto- nated, which renders it unable to maintain stable p53 olig- omers. This structurally subtle and somewhat mild defect is sufficient to cause LFS. The molecular alteration reported here may also represent a relatively mild defect, and it is possible this mutation may, under specific circumstances,
retain a degree of wild-type p53 protein activity. This may explain why the two affected children have developed cancers in infancy, suggesting a highly penetrant mutation, but two other carrier children in the family (aged 3 years and 18 months) have yet to manifest any abnormality, sug- gesting a less penetrant mutation. Thus, the mutation pene- trance may depend upon some other genetic modifiers.
The kindred described here show characteristic genetic anticipation, a relatively common feature among LFS fam- ilies. Tumors are present in the three documented genera- tions, occurring in middle age in the grandmother, in the mid-20s in the mother, and in early childhood in affected children. This observation is compatible with the hypothe- sis that presence of a germline TP53 mutation may affect genetic and genomic stability, favoring the accumulation of genetic damage over successive generations. These addi- tional genetic events may compound the phenotype, which would explain the earlier occurrence of cancer in the youn- ger generation.
Ethical considerations may prevent the practice of screening unaffected children in families with LFS; most
published guidelines recommend postponing testing of at- risk children until after the age of 18 years. This practice is not universal, however, and there are authors who believe that presymptomatic testing in childhood may be beneficial [10,11]. We feel that we should not deny the request of par- ents who wish to screen their presymptomatic children, in that screening can allow the parents to be emotionally and physically prepared in the event of cancer development or, alternatively, can remove unwarranted anxiety if the child is discovered not to be a carrier. In addition, knowl- edge of LFS carrier status helps health personnel identify which individuals require appropriate follow-up for cancer surveillance. In the present case, all children who have been identified to carry the TP53 mutation are being monitored in our pediatric oncology unit. The child with adrenal car- cinoma is seen at 6-month intervals for abdominal ultraso- nography and blood tests. Her parents have been informed that there is a risk of her developing other cancers, as has been reported in patients with LFS [12-14]. The asymp- tomatic carrier children are undergoing yearly clinical examination and screening for adrenal carcinoma using serum cortisol and testosterone. Further blood tests or ra- dioimaging will depend on symptoms or clinical findings. It may be argued that clinical examination and ultrasonog- raphy may not be as precise as whole-body magnetic resonance imaging to detect small malignant lesions. How- ever, we do not recommend whole-body magnetic reso- nance imaging, because cost-effectiveness has not been demonstrated-a key consideration in countries with lim- ited resources. In addition, young children would need to undergo anesthesia for this procedure.
Another potential screening approach using [18F]fluoro- deoxyglucose positron emission tomography-computed tomography has been recently reported in LFS families: 3 of 15 individuals were detected to have asymptomatic thy- roid and esophageal cancer [15]. This modality is expen- sive, however, and involves radiation exposure.
The family described here is, to our knowledge, the first reported case of LFS from Southeast Asia. Previous reports involving Asian populations have been from East Asia (i.e., from Japan [16,17] and Korea [18,19]). Currently, no re- gional LFS database exists, and no information is available on the possible incidence of LFS in countries such as Thai- land, Malaysia, Singapore, and Indonesia (which account for a population of almost 330 million). We have recently started a LFS screening project in Malaysia, where central laboratory facilities are available, initially targeting pediat- ric oncologists. It is hoped that this will serve as a nidus for the dissemination of knowledge on LFS as well as acquisi- tion of information on germline TP53 mutations in South- east Asia.
Acknowledgments
We thank the pediatric oncology team of University Malaya Medical Center who were involved in the clinical
management of this patient and her sibling and Dr. Maria Isabel Achatz for advice on counseling and follow-up. This work was supported by funding from the World Health Organization-International Agency for Research on Cancer and from the Ministry of Higher Education, Malaysia.
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