Three new BLM gene mutations associated with Bloom syndrome

Three New BLM Gene Mutations Associated with Bloom Syndrome

Mounira Amor-Gue´ret,

Catherine Dubois-d’Enghien,

Anthony Lauge´,

Rosine Onclercq-Delic,

Abdelhamid Barakat,

Elbekkay Chadli,

Ahmed Aziz Bousfiha,

Meriem Benjelloun,

Elisabeth Flori,

Be´re´nice Doray,

Vincent Laugel,

Maria Teresa Lourenc¸o,

Rui Gonc¸alves,

Silvia Sousa,

Je´roˆme Couturier,

and Dominique Stoppa-Lyonnet

Bloom’s syndrome (BS) is a rare autosomal recessive disease predisposing patients to all types of cancers affecting the general population. BS cells display a high level of genetic instability, including a 10-fold increase in the rate of sister chromatid exchanges, currently the only objective criterion for BS diagnosis. We have developed a method for screening the BLM gene for mutations based on direct genomic DNA sequencing. A questionnaire based on clinical information, cytogenetic features, and family history was addressed to physicians prescribing BS genetic screening, with the aim of confirming or guiding diagnosis. We report here four BLM gene mutations, three of which have not been described before. Three of the mutations are frameshift mutations, and the fourth is a nonsense mutation. All these mutations introduce a stop codon, and may therefore be considered to have deleterious biological effect. This approach should make it possible to identify new mutations and to correlate them with clinical information.

Introduction

B loom’s syndrome (BS) is a very rare human autosomal recessive disorder characterized by marked genetic in- stability associated with a strong predisposition to a wide range of cancers commonly affecting the general population.

The hallmark of BS cells is their high frequency of sister chromatid exchanges (SCEs), which is currently the only objective criterion for diagnosis of the disease. The two main clinical features of BS are proportionate pre- and postnatal growth retardation and a high frequency of cancers. Addi- tional clinical features include dolichocephaly, facial sun- sensitive telangiectatic erythema, patchy areas of hyper- and hypopigmentation of the skin, and moderate to severe im- munodeficiency, manifested by recurrent respiratory tract and gastrointestinal infections (German, 1993). BS is caused by inactivating mutations in both copies of the BLM gene,

which is located on chromosome 15 at 15q26.1 (Ellis et al., 1995). Nonsense or frameshift mutations leading to the in- troduction of a premature termination codon and missense mutations have been found in the BLM genes of BS patients (Rong et al., 2000; German et al., 2007). One particular BLM gene mutation, corresponding to a 6-bp deletion and a 7-bp insertion at nucleotide position 2207, known as the blm

mutation, has been identified in almost all BS patients of Ashkenasi Jewish ancestry reflecting a founder effect (Ellis et al., 1995). The BLM gene encodes the BLM protein, a 1417 amino acid protein with a predicted molecular weight of 159 kDa. BLM belongs to the DExH box-containing RecQ helicase subfamily (Ellis et al., 1995). Recombinant BLM displays ATP- and Mg

-dependent 3

-5

-DNA helicase activity (Karow et al., 1997). However, the physiological function of BLM remains unclear. Several studies have pro- vided evidence that BLM plays a major role in maintaining

1

Institut Curie, Centre de Recherche, Orsay, France.

2

CNRS, UMR2027, Orsay, France.

3

Institut Curie, Hoˆpital, Service de Ge´ne´tique Oncologique, Paris France.

4

Institut Pasteur du Maroc, Service de Ge´ne´tique, Casablanca, Morocco.

5

CHU Ibn Rochd, Unite´ d’Immunologie Clinique du Service de Pe´diatrie, 1, Casablanca, Morocco.

6

Service de Cytoge´ne´tique, Hoˆpital de Hautepierre, Strasbourg Cedex, France.

7

Service de Ge´ne´tique Me´dicale, Hoˆpital de Hautepierre, Strasbourg Cedex, France.

8

Service de Pe´diatrie 1, CHU Strasbourg-Hautepierre, Strasbourg Cedex, France.

9

Servic¸o de Genetica Me´dica, Hospital de Dona Estefania, Lisboa, Portugal.

10

Servic¸o de Medicina II, Hospital de Egas Moniz, Lisboa, Portugal.

11

INSERM, U380, Paris, France.

ª Mary Ann Liebert, Inc.

Pp. 257–262

DOI: 10.1089=gte.2007.0119

257

genomic stability during DNA replication, recombination, and repair, and particularly in restarting stalled replication forks (Amor-Gue´ret, 2006). We recently developed a muta- tion screening for the BLM gene based on the direct se- quencing of its coding exons and their flanking regions. We identified four BLM gene mutations in four unrelated pa- tients, three of which have not been reported elsewhere.

Materials and Methods

Four patients diagnosed with BS on the basis of typical clinical features (Table 1) and high frequency of SCEs were referred to our laboratory by physicians. Two of the physi- cians contacted us via the Orphanet online genetic services database (http: == www.orpha.net = ). A questionnaire on clin- ical information, cytogenetic features, and family history was drawn up (supplementary data available online at www .liebertpub.com). It was completed by the referring physi- cians for two cases (IC-T1 and IC-P1). All the clinical features of patient IC-IPM2 have been reported in detail in an M.D.

thesis (Benjelloun Meriem, 2005, available upon request).

Clinical information regarding patient IC-I1 were kindly communicated to us by Dr. M. Debre´ and Dr. S. Blanche (Hoˆpital Necker Enfants-Malades, Paris, France). Informed consent for BLM gene analysis was obtained from the pa- tient’s parents by the referring physicians. DNA was ex- tracted from peripheral blood in three cases and from primary fibroblasts in one case (IC-I1). DNA samples from lymphoblastoid BS cell lines derived from a patient bearing a homozygous Ashkenazi BLM mutation and from a Mor- occan patient bearing a homozygous missense mutation were used as controls for BLM genotyping (Ellis et al., 1995;

Barakat et al., 2000). The entire coding sequence of BLM (exons 2 to 22) was analyzed, including all intron–exon junctions and at least 70 bp of the intronic sequences flanking each exon. PCR primers flanking the 21 coding exons of BLM were designed using the Primer 3 program (http:==frodo .wi.mit.edu = cgi-bin = primer3 = primer3_www.cgi) (Table 2).

We avoided intronic sequences containing known polymor- phic sites to prevent false-negatives resulting from the amplification of a single allele (ENSEMBL database). Each fragment was amplified from genomic DNA in a 50 m L re- action volume, using a GeneAmp PCR system 9700 (Applied Biosytems, Foster City, CA). The PCR mixture contained 50 ng DNA, 0.2 mM dNTPs (Applied Biosytems), 0.025 U =m L Thermoprime Plus DNA polymerase (Abgene, Epsom, UK), and 0.3 m M of each forward and reverse primer in reaction buffer (1.5 mM MgCl

, 75 mM Tris-HCl pH 8.8, 20 mM (NH

)

SO

, 0.1% (v = v) Tween 20). Amplification was per- formed with a touchdown PCR program consisting of an initial denaturation step at 948C for 5 min followed by 35 PCR cycles: 30 s of denaturation at 94 8 C, 30 s of anneal- ing with temperature decreasing gradually from 608C to 52 8 C during the 35 cycles, and 30 s of elongation at 72 8 C.

PCR products were separated by agarose gel electrophore- sis, purified (Macherey-Nagel, Du¨ren, Germany), and se- quenced, using BigDye Terminator v1.1 Cycle Sequencing Kit and an ABI 3130xl automated sequencer (Applied Bio- systems). SeqScape

(Applied Biosystems) software was used for sequence analysis. Precisely, we proceed as follow:

we first sequence the BLM gene using forward PCR primers (Table 2); when a mutation is identified with this first screen,

we further sequence the corresponding region using reverse primers (Table 2); when the quality of the first sequence is not sufficient, the reverse sequencing is done on the total 21 PCR products; if no mutation is detected, alternative meth- ods will be used, as developed in the discussion section.

Results

The four patients presented most of the typical clinical fea- tures of BS: short stature, narrow face, sun-sensitive erythema, hyperpigmentation spots on the body, and susceptibility to respiratory tract infections (Table 1). We also observed some clinical features not usually reported in BS. Indeed, two of the three patients for whom head circumference measures were available (IC-T1, IC-P1) presented microcephaly. Another two patients (IC-I1 and IC-IPM2) presented ocular telangiectasia, and one patient (IC-T1) had two right kidneys. None of the patients had cancer at their most recent follow-up visit, at which they were between 1 and 28 years old (Table 1).

The countries of origin of the patients are reported in Table 1. Patient IC-I1 has previously been reported as PuCh (Foucault et al., 1997). Three of the four patients were born to cousins, whereas the parents of IC-I1 were unrelated.

As expected, the two BLM mutations of the controls were detected and found to be homozygous (data not shown).

In addition to the missense mutation (c.3107G > T=p.

Cys1036Phe) previously described in patient IC-I1 (Foucault et al., 1997), we identified the other four expected mutations, three of which were homozygous as predicted by the consan- guinity of their parents (Table 1). These four mutations gen- erated premature termination codons. Three of the mutations were frameshift mutations, and one was a nonsense mutation.

Discussion

We report here four BLM gene mutations identified by the direct sequencing of genomic DNA. All the expected muta- tions were identified, validating our methodological ap- proach. However, direct sequencing can fail to detect large rearrangements of BLM, the frequency of which was esti- mated at 6% based on the recent review by German et al.

(2007). Semi-quantitative PCR methods quantitative multi- plex PCR of shorts fluorescents fragments (QMPSF), multi- plex PCR liquid chromatography (MP = LC), or multiplex ligation-dependent probe amplification (MLPA) must be used when no mutation is detected in a patient identified as having BS on the basis of a high frequency of SCE.

All four mutations reported introduce a stop codon, con- sistent with the frequency of protein-truncating mutations identified in the BLM gene. Indeed, 74 different BLM gene mutations have been reported to date: 81% (60 = 74) introduce a premature termination codon, and 19% (14=74) are mis- sense mutations (http: == www.uta.fi = imt = bioinfo = BLMbase) (Rong et al., 2000; German et al., 2007). Frameshift and non- sense mutations are found along the entire length of the BLM gene, whereas all missense mutations affect the helicase or the RecQ-CT domain, indicating that these domains are crucial for the function of BLM.

One of the mutations identified, 3587delG = p.Ser1196fsX3,

was recently described as a Portuguese = Brazilian mutation,

as it was found in two BS patients from Brazil (German et al.,

2007). Interestingly, this mutation was present in the ho-

mozygous state in the Portuguese BS patient, further sup-

Table 1. Patient Characteristics and BLM Gene Mutations Patients Sex Age

(years) Height

(cm)

Head circumference

(cm) Family

Sun-sensitive erythema

Hype r = hypo- pigmentation spots

Respiratory tract infections Gastro-intestinal infections Immune defect Cancer

Others

BLM gene mutations

Allele 1 Allele 2 IC-T1 M 9 118.5 ( 2.5 SD) 47.5 ( 3.8 SD) Yes Yes Yes Yes No ND

No 2 right kidneys c.581- 582delTT = p.Phe194X c.581- 582delTT = p.Phe194X IC-P1 F 28 142.5 ( 3.6 SD) 50.5 ( 3.8 SD) Yes Yes Yes Yes No IgG IgM No No c.3587 delG = p.Serl196ThrfsX3 c.3587 delG = p.Serl196ThrfsX3 IC-IPM2 F 5 82 ( 6 SD) ND

Yes No Yes Yes Yes IgM No Warts ocular telang

c.850- 873del23 = p.Glu284-Phe291 > X c.850 873del23 = p.Glu284-Phe291 > X IC-I1 F 1 69 ( 2.2 SD) 39 ( 6 SD) No No Yes No No IgM No Ocular telang

c.3107 G > T = p.Cys1036Phe c.2821 C > T = p. Gln941X

At the last follow- up.

Paren t consangui nity.

On the face.

On the body .

Acc ording to the cDNA codi ng seq uence, w ith þ 1 the A o f the initiating A T G .

Not det ermined.

Oc ular tela ngiectas ia.

Previ ously report ed in Fouca ult et al ., 1997.

porting its founder origin. The other three mutations have not been described before, consistent with the considerable diversity of mutations affecting the BLM gene. This diversity contrasts with the very low incidence of BS, varying from 1 in 48,000 in Ashkenazi Jews to 1 in 10,836,000 in Japan (German et al., 1989; Li et al., 1998; Shahrabani-Gargir et al., 1998). Indeed, the diversity of the mutations observed and the length of the coding sequence (4521 bp) suggest that a higher disease incidence would be expected. German found that fewer children than expected were affected in the sib- ships of BS patients (German, 1969). This may be due to the loss of some homozygotes during embryonic or fetal devel- opment. Two of the mothers of patients described here had suffered miscarriages: in one case (IC-P1), the mother suf- fered two late-onset miscarriages, at 24 weeks and 32 weeks of pregnancy. Interestingly, four of the five BS mouse models developed to date display embryonic lethality (Chester et al., 1998; Luo et al., 2000; Goss et al., 2002). The only viable ho- mozygous Blm mouse mutant has actually been shown to carry a hypomorphic Blm allele (McDaniel et al., 2003). How- ever, hypomorphic BLM mutations are unlikely to account for the low frequency of the disease in humans. Indeed, there is no evidence to suggest that a residual functional protein results from truncating mutations. Further, hypomorphic mutations would not result in the observed deficit of affected patients in BS sibships. The very low disease frequency may be due to the effects of other genetic determinants rescuing embryos from lethality. We previously speculated that BLM-deficient cells may escape apoptotic death and survive by constitutively inducing a bacterial SOS-like response (Amor-Gue´ret, 2006).

The capacity to induce such a response may be genetically determined.

Acknowledgments

We are grateful to Dr. S. Blanche and Dr. M. Debre´ (Hoˆ- pital Necker Enfants-Malades, Paris, France) for providing us with IC-I1 clinical data. We thank Patricia Legoix-Ne´ for her

help in DNA sequencing (Institut Curie, Paris, France), the technicians of the Cytogenetic Unit (Strasbourg, France), and Clare Ferreira Pinto (Servic¸o de Genetica Me´dica, Hospital de Egas Moniz, Lisboa, France) for the analysis of SCEs. We also thank the managers of Orphanet for facilitating connections between physicians, researchers, and patients.

This work was supported by funds from the Institut Na- tional du Cancer (Re´seau Pre´dispositions He´re´ditaires aux

« maladies cassantes » de l’ADN), Institut Curie, and the Centre National pour la Recherche Scientifique. M.A.G.

would also like to thank the Director of Institut Curie Re- search Center for supporting her involvement in this project.

References

Amor-Gue´ret M (2006) Bloom syndrome, genomic instability and cancer: the SOS-like hypothesis. Cancer Lett 236:1–12.

Barakat A, Ababou M, Onclercq R, Dutertre S, Chadli E, Hda N, Benslimane A, Amor-Gueret M (2000) Identification of a novel BLM missense mutation (T2706C) in a Moroccan patient with Bloom’s syndrome. Hum Mutat 15:584–585.

Chester N, Kuo F, Kozak C, O’Hara CD, Leder P (1998) Stage-specific apoptosis, developmental delay, and embryonic lethality in mice homozygous for a targeted disruption in the murine Bloom’s syndrome gene. Genes Dev 12:3382–3393.

Ellis NA, Groden J, Ye TZ, Straughen J, Lennon DJ, Ciocci S, Proytcheva M, German J (1995) The Bloom’s syndrome gene product is homologous to RecQ helicases. Cell 83:655–666.

Foucault F, Vaury C, Barakat A, Thibout D, Planchon P, Jaulin C, Praz F, Amor-Gueret M (1997) Characterization of a new BLM mutation associated with a topoisomerase IIa defect in a pa- tient with Bloom’s syndrome. Hum Mol Genet 6:1427–1434.

German J (1969) Bloom’s syndrome. I. Genetical and clinical observations in the first twenty-seven patients. Am J Hum Genet 21:196–227.

German J (1993) Bloom syndrome: a Mendelian prototype of somatic mutational disease. Medicine (Baltimore) 72:393–406.

German J, Takebe H (1989) Bloom’s syndrome. XIV. The disor- der in Japan. Clin Genet 35:93–110.

Table 2. Pairs of Primer Sequences Used for BLM Gene Amplification and Sequencing

Name Forward sequence Reverse sequence

BLM-02 5

-GCCTGTTTGAATCAAGTCC-3

5

-AGCTTCATTTCCTCATCTG-3

BLM-03 5

-TGGATTCTTTGCTCAGTTGG-3

5

-TCATGACTATTCCCAATGGC-3

BLM-04 5

-TCATTCTTAATCGCTCATGC-3

5

-GAGGCTTTCACTTGAATGTA-3

BLM-05 5

-TATTGTCTGATCAGTGGTAG-3

5

-CACAGGTTCAAAACACAATC-3

BLM-06 5

-TTACAGTCATGAGCCACCAT-3

5

-TTTTGCCCTGTCCATTTTTC-3

BLM-07 5

-AGATTTGCTTTTGTGGCCTA-3

5

-CCTATTTTGGGACAGTTTTG-3

BLM-08 5

-AGGGCAAGGGAAATGCTAAA-3

5

-GGTTTAAGAGGTCCCTAAATG-3

BLM-09 5

-TGAGTATGGCAAATTGTTGG-3

5

-TGCAAATTTAACTGCTGTGC-3

BLM-10 5

-CCTTTGATAGGTTTGATATGTGAC-3

5

-TTGGGGTTTCTGGATGAAAG-3

BLM-11 5

-CAGCTTAAGTTGTGATGGAA-3

5

-GTTCACTCAGTGTGGGTTTT-3

BLM-12 5

-AAAACCCACACTGAGTGAAC-3

5

-CCACAGGTGATTCTAACAT-3

BLM-13 5

-GGGGACACATGTAGTCTATAA-3

5

-GCTGTCATAATGCAAAAAGG-3

BLM-14 5

-TATTCACGTGTGTGGTCTTC-3

5

-AGTTTGCATTCTACATGTGC-3

BLM-15 5

-GGCATTGCAAGTTATCAGTA-3

5

-TAAGGAAAACTGGACCAGAA-3

BLM-16 5

-CCATGTTGACAATGCTGTTA-3

5

-AGACCACCTTTTGCAATCTA-3

BLM-17 5

-TCTGGAAATGGGTTATGATG-3

5

-CACTCAGATGAACTCGCATA-3

BLM-18 5

-CCAAACCTGTCCATAAATGA-3

5

-AAGCTTGGACAAAGACACTA-3

BLM-19 5

-TGGGTTATCAGGTCACTAAA-3

5

-AAGGAGTTCCCTAGCAAGAT-3

BLM-20 5

-TGTGCTGAATGCGTGAAT-3

5

-CACACAACTGCATCCTTCTA-3

BLM-21 5

-GCAGCTAGGTATCTGCTAAAA-3

5

-ATCCTTCAAAGCAAGGCAGA-3

BLM-22 5

-ATTGTAGCTCTGTGCAGGTT-3

5

-TCAGACGTTCTGAGAAACAA-3

German J, Sanz MM, Ciocci S, Ye TZ, Ellis NA (2007) Syndrome- causing mutations of the BLM gene in persons in the Bloom’s syndrome registry. Hum Mutat 28:743–753.

Goss KH, Risinger MA, Kordich JJ, Sanz MM, Straughen JE, Slovek LE, Capobianco AJ, German J, Boivin GP, Groden J (2002) Enhanced tumor formation in mice heterozygous for Blm mutation. Science 297:2051–2053.

Karow JK, Chakraverty RK, Hickson ID (1997) The Bloom’s syndrome gene product is a 3

-5

DNA helicase. J Biol Chem 49:464–469.

Li L, Eng C, Desnick RJ, German J, Ellis NA (1998) Carrier fre- quency of the Bloom syndrome blmAsh mutation in the Ashkenazi Jewish population. Mol Genet Metab 64:286–290.

Luo G, Santoro IM, McDaniel LD, Nishijima I, Mills M, Yous- soufian H, Vogel H, Schultz RA, Bradley A (2000) Cancer predisposition caused by elevated mitotic recombination in Bloom mice. Nat Genet 26:424–429.

McDaniel LD, Chester N, Watson M, Borowsky AD, Leder P, Schultz RA (2003) Chromosome instability and tumor pre- disposition inversely correlate with BLM protein levels. DNA Repair (Amst) 2:1387–1404.

Rong SB, Valiaho J, Vihinen M (2000) Structural basis of Bloom syndrome (BS) causing mutations in the BLM helicase domain.

Mol Med 6:155–164.

Shahrabani-Gargir L, Shomrat R, Yaron Y, Orr-Urtreger A, Groden J, Legum C (1998) High frequency of a common Bloom syndrome Ashkenazi mutation among Jews of Polish origin. Genet Test 2:293–296.

Address reprint requests to:

M. Amor-Gue´ret, Ph.D.

Institut Curie, Centre de Recherche UMR 2027 CNRS, Centre Universitaire Baˆtiment 110 91405 Orsay Cedex France E-mail: [email protected]

D. Stoppa-Lyonnet, M.D., Ph.D.

Institut Curie, Hoˆpital

Service de Ge´ne´tique Oncologique

26, rue d’Ulm, 75248

Paris Cedex 05

France

E-mail: [email protected]