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Human Molecular Genetics Pages 69-79  

Characterization of ATM gene mutations in 66 ataxia telangiectasia families
Introduction
Results
Discussion
Materials And Methods
   Patients
   Sequence analysis
   Transcript analysis
   Western blotting
Acknowledgements
References

Characterization of ATM gene mutations in 66 ataxia telangiectasia families

Characterization of ATM gene mutations in 66 ataxia telangiectasia families

Natalia Sandoval, Matthias Platzer, André Rosenthal, Thilo Dörk1,*, Regina Bendix1, Britta Skawran1, Manfred Stuhrmann1, Rolf-Dieter Wegner2, Karl Sperling2, Sharon Banin3, Yosef Shiloh3, Alessandra Baumer4, Ulrike Bernthaler4, Helga Sennefelder4, Monika Brohm4, Bernhard H. F. Weber4, Detlev Schindler4,+

Department of Genome Analysis, Institute of Molecular Biotechnology, Jena, Germany, 1Institute of Human Genetics, Medical School Hannover, D-30625 Hannover, Germany, 2Institute of Human Genetics, Virchow Clinics, Humboldt University, Berlin, Germany, 3Institute of Human Genetics, Sackler School of Medicine, Tel Aviv, Israel and 4Department of Human Genetics, University of Würzburg, Würzburg, Germany

Received July 17, 1998; Revised and Accepted October 13, 1998

Ataxia telangiectasia (AT) is an autosomal recessive disease characterized by neurological and immunological symptoms, radiosensitivity and cancer predisposition. The gene mutated in AT, designated the ATM gene, encodes a large protein kinase with a PI-3 kinase-related domain. In this study, we investigated the mutational spectrum of the ATM gene in a cohort of AT patients living in Germany. We amplified and sequenced all 66 exons and the flanking untranslated regions from genomic DNA of 66 unrelated AT patients. We identified 46 different ATM mutations and 26 sequence polymorphisms and variants scattered throughout the gene. A total of 34 mutations have not been described in other populations. Seven mutations occurred in more than one family, but none of these accounted for more than five alleles in our patient group. The majority of the mutations were truncating, confirming that the absence of full-length ATM protein is the most common molecular basis of AT. Transcript analyses demonstrated single exon skipping as the consequence of most splice site substitutions, but a more complex pattern was observed for two mutations. Immunoblot studies of cell lines carrying ATM missense substitutions or in-frame deletions detected residual ATM protein in four cases. One of these mutations, a valine deletion proximal to the kinase domain, resulted in ATM protein levels >20% of normal in an AT lymphoblastoid cell line. In summary, our results survey and characterize a plethora of variations in the ATM gene identified by exon scanning sequencing and indicate a high diversity of mutations giving rise to AT in a non-isolated population.

INTRODUCTION

Ataxia telangiectasia (AT) is an autosomal recessive disorder that affects ~1:40 000-1:300 000 children in various ethnic groups (1-4). AT is a pleiotropic disease characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, thymus dysplasia, immunodeficiency and bronchopulmonary infections, gonadal dysgenesis, chromosomal instability, abnormal X-ray sensitivity and cancer predisposition (1,2). Cultured cells from AT patients are also hypersensitive to ionizing radiation and show defective activation of radiation-induced cell cycle checkpoints, including retarded p53 stabilization (3-8). Heterozygous carriers of an AT mutation are clinically unaffected, but there is reported evidence from epidemiological studies that AT heterozygosity predisposes to some epithelial cancers, in particular breast cancer (9-13). The gene mutated in AT, ATM, has been localized to chromosome 11q23 (14) and subsequently was isolated and characterized (15,16). It spans ~150 kb of genomic sequence and contains 66 exons (17,18). The ATM gene gives rise to a ubiquitously expressed transcript of ~13 kb which encodes a nuclear 350 kDa protein with homology to PI-3 kinases and related proteins involved in DNA damage responses and cell cycle regulation (16,19-22). While the precise function of the ATM gene is still unknown, the ATM gene product is yet believed to sense DNA double-strand breaks (7) and to regulate physiological responses via p53- or Chk1-mediated pathways (4,23). Furthermore, the non-receptor tyrosine kinase c-Abl has been identified as a downstream target of the ATM protein (4,24,25).

Numerous different mutations in the ATM gene have been identified in classical AT and in some patients with variant forms of AT (26). The majority of the published mutations in the ATM gene are truncating, although missense substitutions and in-frame-deletions have also been found (15,27-36). Until now most studies have used cDNA-based screening techniques for mutation analysis and, thus, a number of the reported exon-skipping mutations have remained uncharacterized at the genomic level (26). Now that the exon-intron structure of the ATM gene has been fully elucidated (17) and complete sequence information is available for the entire ATM gene (18), mutation scanning methods using genomic DNA have become feasible. In the present study, we established primers and PCR conditions to amplify all exons and exon-flanking intron regions of the ATM gene. We applied automated sequencing of the PCR products to mutation scanning. As a result, we report the spectrum of ATM gene mutations identified in 66 AT families diagnosed in Germany.


Figure 1. View of the XGAP editor and selected trace files of an AT sub-project with 18 patient samples.


Figure 2.. Distribution of mutations throughout the ATM gene in a group of 66 AT patients. In-frame or frameshift deletions, frameshift insertions, nonsense, missense and splicing mutations are indicated using different symbols.


RESULTS

We designed a set of 68 PCR primer pairs based on the published genomic sequence information to amplify all 66 exons and the adjacent intron regions of the ATM gene. The primers were arranged in a fashion to allow for amplification of most exons under the same conditions. Due to its large size of 3.8 kb, the last exon 66 was amplified in nine overlapping fragments. In a few other instances, two exons were amplified jointly together with a small intervening sequence. All primers and PCR conditions are compiled in Materials and Methods.

Table 1. Ataxia telangiectasia mutations
Mutation Nucleotide change Location Predicted effect Patient ID (status)
26delT Deletion of T at 26-27 Exon 4 Truncation Aa053 (hom)
72+2T->C T->C at 72+2 Intron 4 Aberrant splicing Aa005 (het)
Y171X T->G at 513 Exon 8 Truncation Aa050 (hom)
788delT Deletion of T at 788-790 Exon 9 Truncation Ae005 (het)
822delT Deletion of T at 822 Exon 9 Truncation Ae009 (hom)
        Aa037 (het)
1561delAG Deletion of 2 bp at 1561-1565 Exon 12 Truncation Aa029 (het)
F570S T->C at 1709 Exon 13 Phe->Ser at 570 Aa003 (het)
2250G->A G->A at 2250 Exon 16 Aberrant splicing Aa033 (het)
        Ac048 (het)
        Ac053 (het)
        Ae006 (het)
R785C C->T at 2353 Exon 17 Arg->Cys at 785 Ac046 (het)
2502insA Insertion of A after 2502 Exon 19 Truncation Aa037 (het)
2565delGins21 Deletion of G and insertion Exon 19 Truncation Ac045 (het)
  of 21 bpa at 2565
2921+3A->T A->T at 2921+3 Intron 21 Aberrant splicing Ae014 (hom)
3085insA Insertion of A after 3085 Exon 23 Truncation Ae007 (het)
3576G->A G->A at 3576 Exon 26 Aberrant splicing Aa025 (hom)
        Aa026 (het)
        Aa040 (hom)
3801delG Deletion of G at 3801-3802 Exon 28 Truncation Aa036 (het)
        Aa039 (hom)
3849delA Deletion of A at 3849-3850 Exon 28 Truncation Aa010 (hom)
4051delT Deletion of T at 4051-4052 Exon 29 Truncation Aa032 (het)
4143insT Insertion of T after 4143 Exon 30 Truncation Aa032 (het)
4630del4 Deletion of 4 bp at 4630-4635 Exon 33 Truncation Aa030 (hom)
4909+1G->A G->A at 4909+1 Intron 34 Aberrant splicing Aa031 (hom)
        Ac044 (hom)
5309delC Deletion of C at 5309 Exon 37 Truncation Aa011 (het)
5441insT Insertion of T after 5441 Exon 38 Truncation Aa006 (het)
5546delT Deletion of T at 5546-5549 Exon 39 Truncation Ae015 (het)
V1913G T->G at 5738 Exon 40 Val->Gly at 1913 Aa021 (het)
5876delA Deletion of A at 5876-5878 Exon 41 Truncation Ae017 (het)
E1978X G->T at 5932 Exon 42 Truncation Aa035 (het)
5977insA Insertion of A after 5977 Exon 42 Truncation Ae010 (het)
E2014X G->T at 6040 Exon 43 Truncation Ae004 (hom)
D2016G A->G at 6047 Exon 43 Asp->Gly at 2016 Aa017 (hom)
R2032K G->A at 6095 Exon 43 Aberrant splicing Aa006 (het)
6096-9del5 Deletion of TTCTT at 6096-9 Intron 43 Aberrant splicing Aa014 (het)
A2067D C->A at 6200 Exon 45 Ala->Asp at 2067 Ae003 (het)
R2227C C->T at 6679 Exon 48 Arg->Cys at 2227 Aa055 (het)
6867insT Insertion of T after 6867 Exon 49 Truncation Aa001 (hom)
R2443X C->T at 7327 Exon 52 Truncation Ae006 (het)
        Ae015 (het)
Y2470D T->G at 7408 Exon 52 Tyr->Asp at 2470 Aa038 (het)
7630-2A->C A->C at 7630-2 Intron 53 Aberrant splicing Aa002 (het)
        Aa009 (het)
        Aa051 (het)
        Ac049 (hom)
7668del4 Deletion of 4 bp at 7668-7674 Exon 54 Truncation Aa028 (hom)
V2662del Deletion of 3 bp at 7983-7991 Exon 56 Deletion of Val at 2662 Ac052 (hom)
8385del10 Deletion of 10 bp at 8385-8404 Exon 59 Truncation Aa005 (het)
R2849P G->C at 8546 Exon 60 Arg->Pro at 2849 Aa034 (het)
G2867R G->C at 8599 Exon 61 Gly->Arg at 2867 Aa027 (het)
8720C->TT C->TT at 8720 Exon 62 Truncation Ae017 (het)
8787-1G->T G->T at 8787-1 Intron 62 Aberrant splicing Aa013 (het)
C2931X T->A at 8793 Exon 63 Truncation Aa002 (het)
8814del11 Deletion of 11 bp at 8814-8824 Exon 63 Truncation Aa020 (het)
Mutations have been named according to the recommended nomenclature of Beaudet and Tsui (58). Nucleotide numbering follows the cDNA sequence reported by Savitsky et al. (16) (GenBank accession no. U33841), with nucleotide position 1 assigned to the first nucleotide of the ATG initiation codon in exon 4. Exon-intron boundaries and numbers are as defined by Uziel et al. (17). Homozygous and heterozygous samples are indicated as `hom' and `het', respectively.
aThe inserted sequence at 2565 in sample Ac045 reads <TACTGATCTAAATAGATCAGT>.

Table 2. Polymorphisms and rare sequence variants of the ATM gene
Nucleotide change Location Allele frequency Amino acid change Restriction site
72+36insAA Intron 4 0.57 None
146C/G Exon 5 0.01 (G) S49C HinfI (+)
496+221T/C Intron 7 0.39 (C) None MspI (+)
901+25T/G Intron 9 0.01 (G) None
901+99C/A Intron 9 0.02 (A) None MnlI (-)
1176C/G Exon 11 0.01 (G) None
1488T/C Exon 12 0.01 (C) None AluI (+)
2148C/G Exon 16 0.01 (G) None MspI (-)
2377-56G/A Intron 17 0.36 (A) None
2572T/C Exon 19 0.02 (C) F858L
3078-77T/C Intron 22 0.37 (C) None MseI (-)
3161C/G Exon 24 0.07 (G) P1054R AlwI (-)
3285-9delT Intron 24 0.18 None
3403-15delA Intron 25 0.37 None
4777-20A/G Intron 33 0.01 (G) None
5320-8T/C Intron 38 0.09 (C) None
5557G/A Exon 39 0.18 (A) D1853N
5558A/T Exon 39 0.03 (T) D1853V MaeIII (+)
6199-61C/G Intron 44 0.09 (G) None
7515+45delTT Intron 52 0.01 None DdeI (-)
7630-123insCAGG Intron 53 0.01 None
8787-56T/C Intron 62 0.39 (C) None MaeII (+)
8850+60G/A Intron 63 0.37 (A) None
10775C/T 3[prime]-UTR 0.32 (T) None
12564T/G 3[prime]-UTR 0.37 (G) None
Sequence alterations and their allele frequencies are listed here as polymorphisms or neutral variants if the variations were found in similar frequencies on control chromosomes or if there was no obvious evidence for a potential disease-causing effect from the nature and location of a rare variation. Some of the variants have previously been described by Vorechovsky et al. (31,62). Restriction enzymes to distinguish between variant alleles are as indicated with (+) designating the creation and (-) the abolition of a recognition site.

A cohort of 66 German AT patients were investigated for their mutations in the ATM gene. From genomic DNA samples of all patients, PCR products were obtained for all exons and exon-flanking intron regions and were sequenced on both strands. We obtained ~25 kb of edited sequence information from each patient (Fig. 1; see also Materials and Methods). A total of 46 different mutations were identified on 75 of the 132 alleles, corresponding to a 57% detection rate. In addition, 26 variants and polymorphisms of unknown biological significance were found. The sequence alterations suggesting pathological consequences (mutations) are compiled in Table 1 and the sequence variants that are likely to represent polymorphisms or neutral variations are compiled in Table 2. The distribution of mutations throughout the ATM gene is illustrated in Figure 2. Each of the mutations was confirmed by an independent screening method in the parental DNA samples whenever available.

A major fraction of 27 different mutations (57%) were nonsense mutations or small frameshift insertions/deletions leading to a premature translation termination codon. This subset of the mutations included 14 frameshift deletions and seven insertions comprising between 1 and 11 nt, as well as one more complex frameshift mutation that had emerged from the deletion of a single guanine and the insertion of a 21 nt quasi-palindromic sequence (Table 1). The truncating mutations were scattered throughout the entire ATM gene without apparent clustering. Our data did not allow us to determine whether differences in the AT phenotype in our patients may result from different locations of the premature termination codon within the ATM gene. At any rate, three termination mutations were located downstream of the deduced kinase domain, indicating that even premature stop codons in the very final part of the protein impair ATM expression, stability or function to a disease-causing degree.

Nine different mutations were identified within splice sites of the ATM gene. Mutations within the conserved AG and GU dinucleotides of the acceptor and donor splice sites were regarded as pathogenic, since this mutation type results in aberrant splicing by exon skipping or activation of cryptic splice sites (42). Both effects were observed to occur together in lymphoblasts from patients with the common mutation 7630-2A->C (Table 3). Another mutation, 2921+3A->T, only affected the poorly conserved position +3 in the donor splice site of intron 21, but nevertheless caused a complex aberrant splicing pattern in ATM mRNA, in which exon 21 alone or both exons 20 and 21 were deleted (Table 3). Three other mutations, 2250G->A, 3576G->A and R2032K substituted the final nucleotide of exons 16, 26 and 43, respectively. All of them were confirmed to represent true splicing mutations since skipping of the affected exon was observed in transcripts from patient lymphoblastoid cell lines or fibroblasts (Table 3). No alternative splicing of these exons was detected in lymphocytes from control persons (43); M. Nicke, personal communication). The families of the three patients with the splicing mutation 3576G->A were of Italian, Turkish and Georgian descent, respectively, suggesting that this splicing mutation may be more common in south or south east Europe than in Germany itself. Finally, a deletion of 5 bp, 6096-9del5, within the polypyrimidine tract of intron 43 was found to be associated with skipping of the downstream exon in fibroblasts of the heteroallelic patient. Since this deletion retains only four pyrimidines immediately preceding exon 44, it apparently impedes splice site recognition and thereby acts as a splicing mutation similar to previously reported polypyrimidine sequence alterations in in vitro reactions and in other genetic diseases (44,45).

Missense substitutions constituted ~20% of the identified mutations. Two missense mutations, R2849P and G2867R, reside within the kinase domain and affect residues that are highly conserved among ATM-homologous proteins from different species (16). No mutations were found within the putative leucine zipper region encoded by exon 27 (16) or within the proposed c-Abl binding site encoded by exon 30 (25). Most missense mutations resulted in the substitution of a conserved amino acid identical in the human and murine ATM proteins, the exception being R785C (46). We screened ~100 control chromosomes from anonymous donors for the presence of missense substitutions identified in the AT patients, in order to exclude the possibility that such amino acid mutations represent common polymorphisms. Five missense substitutions (S49C, F858L, P1054R, D1853V and D1853N) were classified as probable polymorphisms after this analysis, since they were also found in our analysis of control chromosomes (P1054R, D1853V and D1853N) or have been previously reported in DNA samples from random blood donors [e.g. by Vorechovsky et al. (31,62)]. Although P1054R was significantly more frequent in our AT patient group (10/132 chromosomes) than in random donors (4/100 chromosomes), this difference appeared to be due to the association of the relatively common AT splicing mutation 3576G->A with the P1054R substitution on the same allele in our AT patients and, thus, to a linkage disequilibrium. With regard to the remaining missense substitutions listed in Table 1, it must cautiously be noted that the absence of an amino acid substitution in a limited number of controls does not fully prove its causative role in the pathogenesis of AT disease. In order to further characterize the effect of non-truncating mutations, we analysed cell lines carrying ATM missense substitutions or in-frame deletions by immunoblot analysis for their levels of ATM protein expression (Fig. 3 and Table 4). A minority of these mutant cell lines revealed detectable but very low amounts of ATM protein. Only the cell line homozygous for the V2662del mutation exhibited higher ATM levels, which varied from culture to culture: This mutant exhibits levels which range between apparently normal and 20% of the normal level. A typical example is shown in Figure 3 (designated L106), which in this case showed a level of ~41%. This protein is probably not very stable and therefore fluctuates according to physiological conditions. It may be possible, but could not be determined, that it is very unstable in certain tissues. The underlying mutation which deletes one of three consecutive valines in exon 56 was identified in a single homozygous German AT patient. The 7-year-old girl developed ataxia by the age of 3 years. She has remained free of recurrent infections so far, despite laboratory evidence of absent IgA and lowered IgG3 levels. Chromosomal instability was shown by increased bleomycin-induced chromosome breakage rates. Hence, clinical and cytogenetic examinations of this patient reveal features of classic AT and further functional analysis of the mutant ATM protein is in progress to yield insight into the molecular pathogenesis of the disease.


Figure 3. Immunoblot analysis of ATM protein levels in lymphoblastoid cell lines. Equal amounts of total protein were loaded in all lanes. L-40 and NL553, normal cell lines; L-6, an AT cell line homozygous for a stop mutation at position 35 of the ATM protein (49); L-106, L-87 and L-105, AT cell lines from patients included in this study (L-106 is homozygous for the V2662del mutation and corresponds to sample Ac052 in Table 1, L-87 is heterozygous for mutation 2250G->A and corresponds to sample Ac053 in Table 1 and L-105 is from a patient whose mutations remained unknown after sequencing all ATM exons and adjacent intron regions).

Table 3. Effects of splicing mutations on ATM mRNA transcripts
Mutation Location Cell type Observed effects
2250G->A Exon 16 B-LCL Skipping of exon 16
2921+3A->T Intron 21 B-LCL Skipping of exon 21 (90%) and joint skipping of
      exons 20+21 (10%)
3576G->A Exon 26 B-LCL Skipping of exon 26
    Fibroblasts Skipping of exon 26
R2032K Exon 43 Lymphocytes Skipping of exon 43
6096-9del5 Intron 43 Fibroblasts Skipping of exon 44
7630-2A->C Intron 53 B-LCL Skipping of exon 54 (60%) and skipping of the first 11 nt
      of exon 54 (40%)
The effect of splicing mutations was characterized by RT-PCR experiments on total RNA isolated from the listed cell types using the primers given in Table 7. Exon skipping has been observed for all investigated mutations. In addition, one cell line exhibited joint skipping of two neighbouring exons and one exhibited an additional alternatively spliced cDNA species due to the use of a cryptic acceptor splice site.

Table 4. Effects of ATM missense and in-frame splicing mutations on ATM protein levels
Mutation Genotype Location Cell type (linea) Protein level
2250G->A 2250G->A/unknown Exon 16 B-LCL (L87a) Trace (6%)
3576G->A Homozygous Exon 26 B-LCL None detected
      Fibroblasts None detected
D2016G Homozygous Exon 43 B-LCL Trace (4%)
Y2470D Y2470D/unknown Exon 52 Fibroblasts None detected
7630-2A->C Homozygous Intron 53 B-LCL None detected
V2662del Homozygous Exon 56 B-LCL (L106a) Variable, ranging from 20% to apparently normal (see text)
R2849P R2849P/unknown Exon 60 B-LCL None detected
G2867R G2867R/unknown Exon 61 B-LCL Trace (2%)
Cell lines with at least one missense mutation or in-frame deletion of the ATM cDNA were analysed for ATM protein levels by immunoblotting.
aThe designation of lymphoblastoid cell lines refers to the designation of samples shown in Figure 3.


When we examined the clinical features of other patients carrying missense mutations, some differences in their disease phenotypes became apparent. One patient homozygous for the missense mutation D2016G was found to produce trace amounts of ATM protein and presented with a protracted disease course. He does not manifest telangiectasia or immunodeficiency at his current age of 7 years. Two siblings who were heterozygotes for the missense mutation F570S, combined with an unidentified second mutation, also showed mild manifestations of AT. Both died in their mid-thirties, which is considered unusually long survival. Unfortunately, no cells were available to study ATM protein levels in these cases. In contrast, two patients heteroallelic for the missense mutations R2849P and G2867R, each in combination with a non-identified second mutation, both expired at age 17 after the classical AT disease course. They showed no (R2849P) or low (G2867R) detectable amounts of ATM protein, suggesting that carrying one missense allele affecting the kinase region may not be sufficient to lessen the disease manifestations. Another two patients homozygous for the in-frame splicing mutations 3576G->A and 7630-2A->C, respectively, likewise showed severe AT manifestations and had no detectable ATM protein.

Several intragenic dimorphisms were identified as a by-product in the sequence analysis during the course of this study (Table 2), in addition to the known intragenic microsatellite at locus D11S2179 present within the exon 62 PCR product. The allele frequencies of the new dimorphisms are compiled in Table 2. Of these dimorphisms, nine surpassed the frequency of 0.3 for both alleles and could be used to construct combined haplotypes over the gene. For example, the dimorphisms in introns 7, 17, 62 and 63 and in the 3[prime]-UTR were in strong linkage disequilibrium and defined two major haplotypes, suggesting that intragenic recombination within the ATM gene is a rare event. No efforts were made in this study to analyse the allele frequencies of these dimorphisms in the general population, since a previous analysis of another linked marker in intron 48 had not revealed any significant differences between the alleles of healthy, breast cancer or AT individuals in the German population (48). Some of the dimorphisms described in our study can be assayed as natural RFLPs (Table 2), which may be useful for indirect genetic diagnosis within AT families or to study loss of heterozygosity in malignancies.

At the end of our sequencing efforts, we were able to identify 46 different ATM mutations on 75 of 132 AT alleles. None of the detected mutations accounted for more than five alleles, and only seven mutations, namely 822delT, 2250G->A, 3576G->A, 3801delG, 4909+1G->A, R2443X and 7630-2A->C, occurred in more than one apparently unrelated AT family. Some of the identified mutations are also present in other populations. There are 12 mutations in our panel whose independent detection has been reported by other investigators: 822delT, 1561delAG, 2250G->A, 2502insA, 3085insA, 3576G->A, 3801delG, E1978X, R2032K, R2443X, 7630-2A->C and 8814del11 (29,35,36). In contrast, the high incidence of new and individual AT mutations in our cohort of patients demonstrates marked mutational heterogeneity of AT in Germany. Finally, it is notable that the complete AT genotype could only be determined in 24 of our patients (17 homozygotes and seven compound heterozygotes), whereas one of both mutations was uncovered in 26 patients and no ATM mutation was found in 16 patients. Thus, further molecular analyses apart from our automated sequencing approach are required to determine the molecular basis of AT disease in the patient group where the complete ATM genotype could not yet be defined.

DISCUSSION

Although numerous mutations have been previously identified in AT families, the present study is the first which systematically examines the whole coding region and all flanking intron portions of the ATM gene by high throughput automated sequencing of PCR products from genomic DNA in a large cohort of AT patients. This approach provides insight into the mutational heterogeneity of AT in Germany and into the polymorphic variability of the ATM gene. Some previous studies reported either a high frequency of distinct AT mutations or uncovered singular mutations that were common in isolated populations due to founder effects (15,27-36,49,59,60). In our cohort, direct sequencing identified 46 different mutations of the ATM gene in 50 of 66 AT patients. These mutations were well distributed over the whole gene, which appears in contrast to hitherto published studies, where mutations were more frequent in the 3[prime]-region of the gene (26). However, the over-representation of C-terminal ATM gene mutations may be explained by more intense screening for mutations in the initially published 3[prime]-part of the gene (26). None of the mutations in our cohort occurred in more than four families and only 12 of the 46 mutations have been independently published for other populations (29,32,34-36). Hence, common mutations exist in AT, but they seem to be present at only low frequencies in Caucasians. It will be interesting to determine by haplotype comparison whether such mutations have a common origin or whether they represent recurrent mutational events. Those mutations which in our study were present in more than one family shared the same intragenic marker haplotype, as exemplified by the association of the 3576G->A splicing mutation with the missense variant P1054R on the same allele in three families with different population backgrounds. This mutation has also been identified by other investigators in Italian AT patients (35). Furthermore, heterozygosity for the P1054R allele has recently been implicated in the aetiogenesis of breast cancer (61), suggesting that either the amino acid substitution P1054R could have a modifying effect on ATM function or that the associated 3576G->A mutation may predispose to malignancy. The mutations 2250G->A and 3801delG have previously been reported in AT families from the British Isles, suggesting an origin in common with the German AT patients in our study (36). Similarly, the mutations 3085insA, R2032K and 7630-2A->C have recently been identified in Polish patients and may share the same origin with the German families reported here (35). In contrast, the previously reported occurrence of mutation R2443X in African-Americans (35) may reflect an independent mutational event as the underlying nucleotide substitution affects a CpG dinucleotide, known hot spots of mutations in general (50).

More than half of the identified AT mutations were nonsense mutations or small frameshift deletions or insertions. Including splicing mutations, four out of five AT mutations are predicted to severely truncate the ATM protein. This finding is an interesting contrast with the mutational spectrum of ATM in T-PLL patients (51,52), but it confirms the predominance of null mutations in AT, consistent with what has been reported in other studies (26,29). Although there are examples of polymorphic stop codons in other genes, e.g. in BRCA2 (53), truncating mutations would usually be expected to result in the loss of ATM function. This view is corroborated by the observation of significantly reduced amounts or the complete absence of truncated ATM proteins with this type of mutation (20,54,55). The sample of ATM missense mutations in our cohort of AT patients is more difficult to address, because only a few of these affect the highly conserved ATM kinase domain and information about other functional domains of the ATM protein is still limited. Most of the identified amino acid substitutions were not present in a series of control chromosomes, however, their absence in a restricted sample from the general population does not yet prove their physiological significance in causing AT disease. Five amino acid substitutions were classified as probably benign on the basis of their occurrence in the general population, but large case-control studies are required to finally exclude the possibility that these are missense variants predisposing towards disease symptoms. Interestingly, our immunoblot analysis of selected missense mutations and in-frame deletions uncovered one amino acid deletion to be associated with variable ATM protein levels in an AT cell line. In the absence of detailed knowledge about ATM structure and substrates, the identification of such naturally ocurring missense substitutions and deletions can be helpful in elucidating residues of physiological importance and may guide in vitro studies to analyse the processing and function of the ATM protein.

In view of the marked allelic heterogeneity of AT in our patients, no general recommendations concerning screening strategies can presently be deduced from our study. The diversity of many distinct mutations scattered throughout a large gene challenges mutation analysis in AT families and hampers the molecular assessment of AT heterozygosity in other clinical entities, such as breast cancer or leukaemias. In addition, the number of mutations presented here accounts for little more than half of all investigated AT alleles. This detection rate is unexpectedly low given the fact that all exons and adjacent intron regions of the ATM gene were sequenced and carefully evaluated on both strands. Direct sequencing is commonly thought to provide a sensitivity close to 100% in the analysed regions and although some problems with heterozygote detection in automated sequencing are known to impair the identification rate of point mutations, this alone is unlikely to account for missing >40% of the ATM mutations. Another reason would be the ascertainment of patients who have a genetically distinct disease, but genetic heterogeneity also seems unlikely in view of the many heterozygotes in whom ATM gene mutations were identified on one of both alleles. Furthermore, some cell lines from patients with unidentified mutations displayed reduced or absent ATM protein in western blot analyses (as exemplified by sample L105 in Fig. 3). A more plausible explanation thus would be the assumption of mutations outside the analysed region, e.g. promotor mutations or intronic splicing mutations of the type that have already been observed in variant AT (33). It is also possible that gross genomic deletions or large inversions account for a substantial portion of AT alleles and have escaped our PCR-based mutation scanning. Similar observations have been made in other inherited diseases, for example familial hypercholesterolemia and haemophilia A (55,56). Further studies at the mRNA level and a search for gross deletions and genomic rearrangements must therefore follow to characterize the unidentified AT alleles. Such studies will complement our automated exon scanning sequencing approach and finally result in an optimized strategy for ATM gene mutation analysis at the genomic DNA level in AT families and for heterozygote detection in conditions where ATM involvement is considered.

MATERIALS AND METHODS

Patients

AT patients and their family members were ascertained at one of the participating centres in Wuerzburg, Hannover or Berlin. The diagnosis was established on the basis of typical clinical features, such as ataxia, telangiectasia, elevated [alpha]-fetoprotein levels and altered immunoglobulin profiles, and was confirmed by laboratory evidence for X-ray or bleomycin sensitivity, such as cytogenetic analysis of chromosome breakage rates or radio-resistant DNA synthesis (37) or by flow cytometric analysis of cell cycle abnormalities after specific induction (38). More detailed information on the clinical and laboratory features of patients with particular ATM genotypes can be obtained from the authors upon request. Only apparently unrelated AT patients were included into this study. Four AT patients in our study have been investigated in parallel using cDNA-based mutation screening and the results have been published elsewhere (27). The genotypes of these patients (designated Aa009, Aa025, Aa027 and Aa028 in Table 1) were resolved at the genomic level in the present study and are consistent with the results obtained by Baumer et al. (27). Little more than half of our 66 AT patients were of German descent, whereas the others originated from Turkey, Italy, Denmark, Switzerland, the Czech Republic, Romania, Georgia or Lithuania. After informed consent had been obtained, blood samples were collected from the AT patients and, wherever available, their parents. Genomic DNA was extracted from white blood cells according to standard procedures. In some cases, established lymphoblastoid or fibroblast cell lines were used as the source for the extraction of genomic DNA.

Table 5. Primers used for genomic PCR amplification
Exon Forward primer Reverse primer Annealing (°C) Size (bp)
1a/1b GGTAGCTGCGTGGCTAACGGAG CCACATACATTATCATCCACTGTAG 59 658
1b/2 AGATAATCTTGACCTGTGGTGAG TCACTAATCTCTTCAAACCTCAG 59 557
3 TTGATTGGCACCAGTTAGTTCAG AGAAGATGCTCATTCACTGATAG 59 303
4+5 TGACCAGAATGTGCCTCTAATTG CACACAAAAGTAATATCACAACAG 59 521
6 ATGTAGTAATCTAAGCAAGGTGG ACAAACTTATGCAACAGTTAAGTC 59 376
7 AAATAGTTGCCATTCCAAGTGTC GATGGAGATGGTATTTTTCAACTG 59 491
8 CAGCAGTTTATAGTTATTCCTGTC TAGTTCTGTTATGATGGATCAATG 59 502
9 CTTTCAGCATACCACTTCATAAC CATAAGTAGCTCCTAGAGGGAAC 54 601
10 TTCTAACGCTGATGCAGCTTGAC CTGATAGTTTGAAAGGAATAGTTG 54 411
11 TGAGTTCCCTGTATCTTCATTTC GTTGAGATGAAAGGATTCCACTG 54 674
12 CTATGGAAATGATGGTGATTCTC GCATCTGAAATAGAATTTGACATC 59 621
13 CTGTCCTGATAGATAAAGTCTTTG TATAAAATAAAGCCATCTGGCATC 59 357
14 ATGGTTGTCCTCCTTAAATTGTC AAACAACCTCTTCCCTGGCTAAC 54 429
15 TAAGGCAAAGCATTAGGTACTTG TTTCTCCTTCCTAACAGTTTACC 54 351
16 GCCACCTTTAACTCAGTTTAACTG CCTGCATAAAATTTAATGTCTTCC 54 354
17 TGAGTTTTGCTTATACTGTATGAC ACTCAGATTCCTATTGCTTATTTG 54 979
18 ATCTACATTCCATTCAAGATAGAG ATGTTAAGAGCTATATGTTGTGAG 59 296
19 GTACTGTAGTTTTCATACTTACAC GGAACAATCCTAAAAGGCTATAC 59 735
20 CAGGTAGTTGCTTTTCTTGATAG ATTACATTTAGTCAGCAACATCAG 59 632
21+22 GTAAATGATTTGTGGATAAACCTG GAAGAAATCACTGATGTGGATAC 59 480
23+24 AGCACAGAAAGACATATTGGAAG ACTATGTAAGACATTCTACTGCC 59 495
25 GTATGTTATTATGTCTCACAGAGTG CTCTCTTATACCAAACTTGGTGAAG 59 378
26 CTGAAATGTGTATAGCTTGTC CACAGTGACCTAAGGAAGCTTC 59 331
27 AATGTTGTGTCTAGGTCCTACTC TGCTTCTCATACTATGTAGGCTC 59 406
28 AGTATGATACTTTAATGCTGATGG AGGTTATATCTCATATCATTCAGG 59 414
29 CCTTTTGAGCTGTCTTGACGTTC GAAATAGACATTGAAGGTGTCAAC 59 271
30 TCTGGACTGTGATATGTCATTTG CTAAAGTGTCACAAGATTCTGTTC 59 513
31 GTGTATTTATTGTAGCCGAGTATC GAAGAACAGGATAGAAAGACTGC 59 315
32 CTGAACAAAAGGACTTCTGAATG ACACTCAAATCCTTCTAACAATAC 54 334
33 CTGGGTATCTTAGACGTAATTAG GCTAGAGCATTACAGATTTTTGAA 59 379
34 GTAGTAATAGAGACATGAGTCAG GACAATGAAACCAAGAGCAAGAC 59 344
35 GGAAGTTCAGATCATTCCCTAC CTATACTGAATACTACAGGCAAC 54 380
36 TATGTATGATCTCTTACCTATGAC CTGAGAATATCTATTTGTGTGAAG 59 302
37 AGTTGCAGTGATTAGTAATTCAAG CAGAACTGTTTTAGATATGCTGG 59 400
38 TTGCCATACCACTCTGCCTCTTG GAAGATGATGTGCAGTATCACAG 59 447
39 ATTCTGTTTCATTATGGTAATGGC TTCCATCTTAAATCCATCTTTCTC 59 529
40 TATCATATTGTGTTGTAATGTCCC ATCATGTTAAAATTCAGCCGATAG 59 524
41 GTCACTACCATTGTATTCTATATC TATACCCTTATTGAGACAATGCC 59 310
42 TGTATTCAGGAGCTTCCAAATAG GCTTAGTCCAGTAAGTAAATTCAG 54 269
43+44 TAGAGTTGGGAGTTACATATTGG TACTGAAATAACCTCAGCACTAC 59 456
45 GGATTTTAAATGATATTGTGAAC CAACTCCTGTTATATTCATAGAAG 54 365
46 AGAGCATATTTAGAACCAGGCAG CTCTTCATCAATGCAAATCCTTAC 59 439
47 GTAGCAAAGCCTATGATGAGAAC TATTGGTAACAGAAAAGCTGCAC 54 279
48 ACAAGTTCTAGTCTTGTCACTAC TAATGACATTTCTTTTTCCCTCAG 59 466
49 GTTAAGTCCTCAATGAATGGTAG CATGATGGACTGATAGAATTGTG 59 479
50 TTGTATGGCAAAAGCAGATGAGG TGAAGCATATTCATGCTAAGTAAC 59 466
51 GAAAATGTACGAATTTGTGTTGGG AGAACCAAGTCACTCTTTCTATG 59 513
52 TTGTAGTTCTGTTAAAGTTCATGG CAAGTGCTAGGAATACAAAGAGG 54 369
53+54 ACTTACTTGCTTAGATGTGAG GGAAAGACTGAATATCACACTTC 59 718
55 GACTGTTTTGTTTGTATCTGAGG TCTACAGAGAGTAACACAGCAAG 59 341
56 CTGCTGACTATTCCTGCTTGACC GCCAATATTTAACCAATTTTGACC 59 253
57 TTCTTAACCACTATCACATCGTC GCATTTCTACTCTACAAATCTTCC 59 405
58 TGAGTGCCCTTTGCTATTCTCAG AGACTCCTGGTCCAAATAATGGC 59 597
59 ATGCTTTGCACTGACTCTGATAG CTTAGTATCTTTGACAATTACCTG 54 367
60 GTAGGTAATGTATCCTGTTCATC AGAATGTAGAAAAAGTGCTGAATC 54 377
61 TATTAGAAAGAGATGGAATCAGTG GGCAAACAACATTCCATGATGAC 59 210
62 AAAGTTCACATTCTAACTGGAAAG GCACTGGAATACGATTCTAGCAC 59 467
63 TATTAAGCATAGGCTCAGCATAC TGCTCTTCACATCAGTGACTTCC 59 257
64 GGTTCTACTGTTTCTAAGTATGTG CCCTACTTAAAGTATGTTGGCAG 54 587
65.1 ATGAATTACCCTTTCATTCAGCC GTAACTACACTAACTGTGTCTTTG 54 630
65.2 ATTTTTCCTAGTAAGATCACTCAG TGGATGAACAGCCTATCAAATAC 59 464
65.3 ACTTGATAGACACTGTAATAGTTC ATCTATATGGGGAGCAAAGAACC 59 454
65.4 CTTCCATGTGTCCCACCTTTATG AACAGAACACAGAGAAATGCTGG 59 600
65.5 CCTTTCTTGTAAGTTCTGCTATG CTTCAAGGTCCAAATACGAGGAG 59 311
65.6 CCTTAGGAAAATGTTCATCCCAG TGCAACAACTAGCAAGAATTAGC 59 479
65.7 TGAAATCTTCATGGGTGAAATTAG ATTGGGATAGTATCACTAATCTTG 54 547
65.8 GTTGTATGCTAAGTCACTGACCC TACTTGAAATTGGGACACATCTC 59 571
65.9 TAAAATGTATGCACTTAGGAATG ACTAGGATATTTAGACTGAGATG 59 497

Table 6. Internal sequencing primers
Exon Designation Sequence
1b s1f2 TCACTCCATCTTTCCTG
1b s1r2 AGGAGGAGGTTATTGGC
2 s1cf3 AGATAATCTTGACCTGTGGTGAG
6 s6r2 CTCACGCGACAGTAATCTG
8 s8f4 TTCTCTGTGTACTTCAGG
11 s11f2 AATGAAGATACCAGATCC
11 s11r2 GGTAGACAAATGACTTAGTTCTG
12 s12f2 TTTTAGGCTACAGATTGC
12 s12r2 GTAAGTCAGACATAATGC
17 s17f4 TCCAAGATCAAAGTACAC
17 s17r1 ATGAGTTGTGACAATCCC
19 s19f1 GATTAGGTAAATTTTGACTACAG
20 s20f2 GCTGTTGTGCCCTTCTCTTAGTG
21 s21r1 CTTAACAGAACACATCAG
22 s22f2 ACCACAGCAATGTGTGTTC
26 s26f5 TTAAGTCCCATAGTGCTG
26 s26r5 TGAGGTTCTAATCCATTC
39 s39f1 GAAATTTAATATGTCAACGGG
45 s45r2 CAAGTCAAATTTCTTACCTG
47 s47r4 GCTGCACTTTAGGATAAC
48 s48f2 TCTTGCTTACATGAACTC
49 s49f4 CAGTAGTAAAAGTATTTATTCCC
53 s53f2 AGGCATACACGCTCTAC
53 s53r2 ATGCCTGCATGTGTGAG
58 s58r1 ACTTCACCCAACCAAATG
62 s62r2 CTCCTTTACTTCATATCAC
65 s64r1 CATATACTGAAGATCACAC

Sequence analysis

PCR conditions were established to separately amplify all exons and exon-flanking intron sequences of the ATM gene for every patient sample (Table 5). PCR amplifications were performed in 50 µl reaction volumes containing 100 ng of genomic DNA, 0.2 mM each dNTP and 2 U Taq DNA polymerase in the reaction buffer supplied by the manufacturer (Boehringer Mannheim). Primer concentrations were 0.5 µM each. As the standard procedure, 35 PCR cycles were performed with 1 min denaturation at 94°C, 1 min annealing at 54 or 59°C, depending on the primer pair, and 1 min elongation at 72°C. The PCR products were purified by PEG precipitation according to a previously published protocol (39) and sequenced on both strands using dye-terminator chemistry (Perkin Elmer). Whenever possible, the PCR primers served as sequencing primers. Because of some background in the sequence trace files or due to poly(A) or poly(T) stretches, 34 sequencing primers had to be chosen anew. These sequencing primers are compiled in Table 6. Sequence information for all exons was generated and filed using ABI 377 automated sequencers. This data was then assembled using the XGAP program (40). To facilitate editing, four XGAP sub-projects were generated, each of which consisted of a backbone of reference sequences derived from genomic clones and the data from 18 patients (Fig. 1). A second round of editing served as an independent confirmation of identified sequence changes.

Transcript analysis

Total RNA was extracted from white blood cells, fibroblasts or lymphoblastoid cell lines by lysis of the cells with guanidinium thiocyanate and extraction of the RNA with acid phenol/chloroform according to the protocol of Chomczynski and Sacchi (41). An aliquot of 1 µg of total RNA was reverse transcribed using a First Strand cDNA Synthesis Kit (Pharmacia) with random hexamers as primers. One-fifth of the cDNA was used as the template in the subsequent PCR amplification of a cDNA region spanning several exons and containing the mutation of interest. The primers used for RT-PCR are compiled in Table 7. Splicing products were analysed after 32 cycles of PCR by electrophoresis in NuSieve agarose, excised and eluted from the gel and sequenced. For quantitative determination of the relative frequencies of the splicing products, the PCR products were radioactively labelled and separated on a 6% denaturing polyacrylamide gel. The incorporated radioactivity was then quantitated by phosphorimager analysis (Fuji).

Western blotting

Protein extraction and immunoblotting analysis were performed as described by Ziv et al. (55) using a monoclonal antibody, ATM 132, raised against positions 819-844 (L. Moyal et al., in preparation).

ACKNOWLEDGEMENTS

We cordially thank the AT families who took part in this study. We are indebted to our clinical colleagues at the different centres who made blood samples from and clinical information about their patients available: Dr I. Bühring and Prof. Dr S. Zielen (Frankfurt), Prof. Dr H.J. Christen (Göttingen), PD Dr J. Freihorst (Hannover), Prof. Dr J. Eggert (Munich), PD Dr O. Riess (Bochum), Dr H. Rickes (Osnabrück), Dr R. Armbrust (Meschede), Prof. Dr E. Seemanova (Prague), Prof. Dr P. Maraschio (Pavia) and Prof. Dr A. Schinzel (Zurich). We cordially thank Nicole Steinlein, Doreen Stoetzer, Diana Wiedemann and Gitta Emmert for expert technical assistance. We are grateful to Prof. Dr J. Schmidtke, Prof. Dr J.H. Karstens and Prof. Dr G. Maass for their kind help and support of this study. Special thanks are due to Dr Michael Oelgeschlaeger for his help in quantitative ATM mRNA evaluation, and to Marion Nicke for additional studies of ATM mRNA in cells from control individuals. Part of the work of the German ATM Consortium has been supported by a generous donation of enzymes from Boehringer Mannheim.

Table 7. Primers used for cDNA analysis
Exons Forward primer Reverse primer
15-19 CAGACAACTTTTGACAAGATGGAC GGTACTTTGGCTCTCTCCAGGTTC
19-22 AGGTGGAGGATCAGTCATCCATG CATCCCTTGTGTTCTCAGAGTCC
22-28 GACTCTGAGAACACAAGGGATGCTC GCATATCATAGACCTTGGTAGCAG
42-45 GCATTTGAAGAAGGAAGCCAG CTTCTAGTTCAGGACACCAGTC
52-55 CGCTTCTATGTAAAGCAGTTG GTCTTCCACTGAGTGGCATC

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