Ataxia-telangiectasia gene and its genomic organization

A purified and isolated gene, designated ATM, mutations of which cause ataxia-telangiectasia, its genomic organization, methods for the detection of the defective gene, the purified polypeptide encoded by the defective gene, and antibodies recognizing the defective protein.

TECHNICAL FIELD
 The present invention relates to the determination of the gene sequence,
 mutations of which cause ataxia-telangiectasia (A-T), designated ATM, and
 the use of the gene and gene products in detection of carriers of the A-T
 gene, and preparing native and transgenic organisms in which the gene
 products encoded by the ATM gene or its homolog in other species are
 artificially produced, or the expression of the native ATM gene is
 modified.
 BACKGROUND OF THE INVENTION
 Ataxia-telangiectasia (A-T) is a progressive genetic disorder affecting the
 central nervous and immune systems, and involving chromosomal instability,
 cancer predisposition, radiation sensitivity, and cell cycle
 abnormalities. Studies of the cellular phenotype of A-T have pointed to a
 defect in a putative system that processes a specific type of DNA damage
 and initiates a signal transduction pathway controlling cell cycle
 progression and repair. For a general review of Ataxia-telangiectasia,
 reference is hereby made to the review Ataxia-Telangiectasis: Closer to
 Unraveling the Mystery, Eur. J. Hum. Genet. (Shiloh, 1995) which, along
 with its cited references, is hereby incorporated by reference as well as
 to the reviews by Harnden (1994) and Taylor et al (1994).
 Despite extensive investigation over the last two decades, A-T has remained
 a clinical and molecular enigma. A-T is a multi-system disease inherited
 in an autosomal recessive manner, with an average worldwide frequency of
 1:40,000-1:100,000 live births and an estimated carrier frequency of 1% in
 the American population. Notable concentrations of A-T patients outside
 the United States are in Turkey, Italy and Israel. Israeli A-T patients
 are Moroccan Jews, Palestinian Arabs, Bedouins and Druzes.
 Cerebellar ataxia that gradually develops into general motor dysfunction is
 the first clinical hallmark and results from progressive loss of Purkinje
 cells in the cerebellum. Oculocutaneous telangiectasia (dilation of blood
 vessels) develops in the bulbar conjunctiva and facial skin, and is later
 accompanied by graying of the hair and atrophic changes in the skin. The
 co-occurrence of cerebellar ataxia and telangiectases in the conjunctivae
 and occasionally on the facial skin--the second early hallmark of the
 disease--usually establishes the differential diagnosis of A-T from other
 cerebellar ataxias. Somatic growth is retarded in most patients, and
 ovarian dysgenesis is typical for female patients. Among occasional
 endocrine abnormalities, insulin-resistant diabetes is predominant, and
 serum levels of alpha-fetoprotein and carcinoembryonic antigen are
 elevated. The thymus is either absent or vestigial, and other
 immunological defects include reduced levels of serum IgA, IgE or IgG2,
 peripheral lymphopenia, and reduced responses to viral antigens and
 allogeneic cells, that cause many patients to suffer from recurrent
 sinopulmonary infections.
 Cancer predisposition in A-T is striking: 38% of patients develop
 malignancies, mainly lymphoreticular neoplasms and leukemias. But, A-T
 patients manifest acute radiosensitivity and must be treated with reduced
 radiation doses, and not with radiomimetic chemotherapy. The most common
 cause of death in A-T, typically during the second or third decade of
 life, is sinopulmonary infections with or without malignancy.
 The complexity of the disease is reflected also in the cellular phenotype.
 Chromosomal instability is expressed as increased chromosomal breakage and
 the appearance in lymphocytes of clonal translocations specifically
 involving the loci of the immune system genes. Such clones may later
 become predominant when a lymphoreticular malignancy appears. Primary
 fibroblast lines from A-T patients show accelerated senescence, increased
 demand for certain growth factors, and defective cytoskeletal structure.
 Most notable is the abnormal response of A-T cells to ionizing radiation
 and certain radiomimetic chemicals. While hypersensitive to the cytotoxic
 and clastogenic effects of these agents, DNA synthesis is inhibited by
 these agents to a lesser extent than in normal cells. The concomitant lack
 of radiation-induced cell cycle delay and reduction of radiation-induced
 elevation of p53 protein are evidence of defective checkpoints at the G1,
 S and G2 phases of the cell cycle. The G1 and G2 checkpoint defects are
 evident as reduced delay in cell cycle progression following treatment
 with ionizing radiation or radiomimetic chemicals, while the rise in the
 p53 protein level usually associated in normal cells with
 radiation-induced GI arrest is delayed in A-T cells. The defective
 checkpoint at the S phase is readily observed as radioresistant DNA
 synthesis (RDS). Increased intrachromosomal recombination in A-T cells was
 also noted recently. Cellular sensitivity to DNA damaging agents and RDS
 are usually considered an integral part of the A-T phenotype. Although
 these clinical and cellular features are considered common to all
 "classical" A-T patients, variations have been noted. Milder forms of the
 disease with later onset, slower clinical progression, reduced
 radiosensitivity and occasional absence of RDS have been described in
 several ethnic groups (Fiorilli, 1985; Taylor et al., 1987; Ziv et al.,
 1989; Chessa et al., 1992). Additional phenotypic variability possibly
 related to A-T is suggested by several disorders that show "partial A-T
 phenotype" with varying combinations of ataxia, immunodeficiency and
 chromosomal instability without telangiectases (12-16) (Ying & Decoteau,
 1983; Byrne et al., 1984; Aicardi et al., 1988; Maserati et a;., 1988;
 Friedman & Weitberg, 1993). Still, other disorders display the A-T
 phenotype and additional features; most notable is the Nijmegen breakage
 syndrome that combines A-T features with microcephaly, sometimes with
 mental retardation, but without telangiectases (Weemaes et al., 1994).
 Prenatal diagnoses of A-T using cytogenetic analysis or measurements of DNA
 synthesis have been reported, but these tests are laborious and subject to
 background fluctuations and, therefore, not widely used.
 A-T homozygotes have two defective copies of the A-T gene and are affected
 with the disease. A-T heterozygotes (carriers) have one normal copy of the
 gene and one defective copy of the gene and are generally healthy. When
 two carriers have children, there is a 25% risk in every pregnancy of
 giving birth to an A-T affected child.
 A-T heterozygotes show a significant excess of various is malignancies,
 with a 3- to 4-fold increased risk for all cancers between the ages of 20
 and 80, and a 5-fold increased risk of breast cancer in women. These
 observations turn A-T into a public health problem and add an important
 dimension to A-T research, particularly to heterozygote identification.
 Cultured cells from A-T heterozygotes indeed show an intermediate degree
 of X-ray sensitivity, but the difference from normal cells is not always
 large enough to warrant using this criterion as a laboratory assay for
 carrier detection. The main reason for the unreliability of this assay is
 the various degrees of overlap between A-T heterozygotes and
 non-heterozygotes with respect to radiosensitivity. Cytogenetic assays for
 carriers have the same problems as for prenatal diagnosis, they are labor
 intensive and not always consistent.
 The nature of the protein missing in A-T is unknown.
 Cell fusion studies have established four complementation groups in A-T,
 designated A, C, D and E, suggesting the probable involvement of at least
 four genes or four types of mutations in one gene, with inter-allelic
 complementation. These four groups are clinically indistinguishable and
 were found to account for 55%, 28%, 14% and 3% of some 80 patients typed
 to date. In Israel, several Moroccan Jewish patients were assigned to
 group C, while Palestinian Arab patients were assigned to group A.
 The general chromosomal localization of the putative A-T gene(s) has been
 determined, but not the sequence. An A-T locus containing the A-T(A)
 mutations was localized by Gatti et al. (1988) to chromosome 11, region
 q22-23, using linkage analysis. The A-T(C) locus was localized by
 applicant to the same region of chromosome 11, region q22-23, by linkage
 analysis of an extended Jewish Moroccan A-T family (Ziv et al., 1991).
 Further studies, conducted by an international consortium in which
 applicant participated (McConville et al., 1990; Foroud et al., 1991; Ziv
 et al., 1992), reconfirmed this localization in a series of studies and
 gradually narrowed the A-T locus to an interval estimated at 4
 centimorgan, which probably contains also the A-T(E) mutations.
 A proposed gene for complementation group D is disclosed in U.S. Pat. No.
 5,395,767 to Murnane et al., issued Mar. 7, 1995. This sequence was found
 not to be mutated in any complementation group of A-T. Further, the gene
 sequence was mapped physically distant from the presumptive A-T locus.
 Therefore, in order to better understand the nature and effects of A-T, as
 well as to more accurately and consistently determine those individuals
 who may carry the defective gene for A-T, it would be advantageous to
 isolate and determine the gene sequence, mutations of which are
 responsible for causing A-T, and utilize this sequence as a basis for
 detecting carriers of A-T and thereby be able to more beneficially manage
 the underlying conditions and predispositions of those carriers of the
 defective gene.
 SUMMARY OF THE INVENTION AND ADVANTAGES
 According to the present invention, a gene, designated ATM, mutations of
 which cause ataxia-telangiectasia (A-T) has been purified, isolated and
 sequenced, as well as mutations of the gene and the genomic organization
 of the gene has been determined.
 The present invention further includes the method for identifying carriers
 of the defective A-T gene in a population and defective A-T gene products.
 The role of the ATM gene in cancer predisposition makes this gene an
 important target for screening. The detection of A-T mutation carriers is
 particularly significant in light of their radiation-sensitivity so that
 carrier exposure to radiation can be properly monitored and avoided.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
 The present invention consists of a purified, isolated and cloned nucleic
 acid sequence (SEQ ID Nos:2, 8-10, 11-63) encoding a gene, designated ATM,
 mutations in which cause ataxia-telangiectasia and genetic polymorphisms
 thereof. The nucleic acid can be genomic DNA, cDNA or mRNA.
 The complete coding sequence of the ATM gene is set forth in SEQ ID No:2
 and was submitted to the GenBank database under accession number U33841.
 There is extensive alternate splicing at the 5' untranslated region
 (5'UTR) of the ATM transcript giving rise to twelve different 5' UTRs. The
 sequence of the longest 5'UTR is set forth in SEQ ID No:9. The first exon
 in this sequence is designated 1b. There is an alternative leader exon,
 designated 1a (SEQ ID No:10). The sequence of the complete 3'UTR is set
 forth in SEQ ID No:8. Together these sequences contain the complete
 sequence of the ATM transcript. The genomic sequence for each exon and
 flanking intronic sequences are set forth in Tables 1 and 5, and SEQ ID
 Nos:11-63.
 As shown in Example 4, using long-distance PCR, the genomic organization,
 i.e. structure, of this gene was determined and the exon-intron boundaries
 identified. The ATM gene spans approximately 150 kb of genomic DNA and
 consists of 66 (64 plus two alternative exon 1) exons. The initiation
 codon falls within the fifth exon. The last exon is 3.6 kb long and
 contains the stop codon and a 3' untranslated region of about 3800
 nucleotides.
 The ATM gene is composed of 66 exons (FIG. 3 and Tables 1 and 5). The first
 two exons are alternatively spliced, and are designated 1a (SEQ ID No:10)
 and 1b (SEQ ID No:9). With the exception of the 3' exon, ATM exons (SEQ ID
 Nos:11-63) range in size from 64 to 372 bp, with an average of 149 bp. The
 introns vary considerably in size, from 100 bp to about 11 kb, with the
 majority in the range of 1-3 kb. The consensus dinucleotides GT and AG
 were found at the donor and acceptor splice sites of all introns, except
 for a variant donor site with a GC dinucleotide (reviewed in Jackson,
 1991) present in the intron 3' to exon 52. The first methionine of the
 open reading frame is located in exon 4, whereas the stop codon is located
 in the 3' and largest exon of 3.6 kb (Exon 65; SEQ ID No:63). This exon
 includes a 3' untranslated region (UTR) (SEQ ID No:8) of about 3800
 nucleotides.
 Polymorphisms are variants in the sequence generally found between
 different ethnic and geographic locations which, while having a different
 sequence, produce functionally equivalent gene products.
 Current mutation data (as shown in Tables 2 and 3) indicate that A-T is a
 disease characterized by considerable allelic heterogeneity. Mutations
 imparting defects into the A-T gene can be point mutations, deletions or
 insertions. The mutations can be present within the nucleotide sequence of
 either/or both alleles of the ATM gene such that the resulting amino acid
 sequence of the ATM protein product is altered in one or both copies of
 the gene product; when present in both copies imparting
 ataxia-telangiectasia. Alternatively, a mutation event selected from the
 group consisting of point mutations, deletions, insertions and
 rearrangements could have occurred within the flanking sequences and/or
 regulatory sequences of ATM such that regulation of ATM is altered
 imparting ataxia-telangiectasia.
 Table 2 illustrates ten mutations in the ATM gene found in A-T patients.
 Mutations in the ATM gene were found in all of the complementation groups
 suggesting that ATM is the sole gene responsible for all A-T cases.
 Table 3 and FIG. 4 illustrate 54 mutations identified to date in
 applicant's patient cohort and include 44 new ones and 10 previously
 listed in Table 2. These mutations were found amongst 55 A-T families:
 many are unique to a single family, while others are shared by several
 families, most notably the 4 nt deletion, 7517del4, which is common to 6
 A-T families from South-Central Italy. The nature and location of A-T
 mutations, as set forth in Table 3, provide insight into the function of
 the ATM protein and the molecular basis of this pleiotropic disease.
 This series of 54 A-T mutations is dominated by deletions and insertions.
 The smaller ones, of less than 12 nt, reflect identical sequence
 alterations in genomic DNA. Deletions spanning larger segments of the ATM
 transcript were found to reflect exon skipping, not corresponding genomic
 deletions. Of the 54 A-T mutations identified, 45 (83.3%) are expected to
 inactivate the ATM protein by truncating it, by abolishing correct
 initiation or termination of translation, or by deleting large segments.
 Additional mutations are four smaller in-frame deletions and insertions,
 and one substitution of a highly conserved amino acid at the PI 3-kinase
 domain. The emerging profile of mutations causing. A-T is thus dominated
 by those expected to completely inactivate the ATM protein. ATM mutations
 with milder effects appear to result in phenotypes related, but not
 identical, to A-T. In view of the pleiotropic nature of the ATM gene, the
 range of phenotypes associated with various ATM genotypes may be even
 broader, and include mild progressive conditions not always defined as
 clear clinical entities as discussed herein below in Example 3. Screening
 for mutations in this gene in such cases will reveal wider boundaries for
 the molecular pathology associated with the ATM gene. The present
 invention therefore allows the identification of these mutations in
 subjects with related phenotypes to A-T.
 The ATM gene leaves a great deal of room for mutations: it encodes a large
 transcript. The variety of mutations identified in this study indeed
 indicates a rich mutation repertoire. Despite this wealth of mutations,
 their structural characteristics point to a definite bias towards those
 that inactivate or eliminate the ATM protein. The nature or distribution
 of the genomic deletions among these. mutations do not suggest a special
 preponderance of the ATM gene for such mutations, such as that of the
 dystrophin (Anderson and Kunkel, 1992) or steroid sulfatase (Ballabio et
 al., 1989) genes which are particularly prone to such deletions. Thus, one
 would have expected also a strong representation of missense mutations,
 which usually constitute a significant portion of the molecular lesions in
 many disease genes (Cooper and Krawczak, 1993; Sommer, 1995). However,
 only two such mutations were identified in the present study. Other point
 mutations reflected in this series are those that probably underlie the
 exon skipping deletions observed in many patients, again, exerting a
 severe structural effect on the ATM protein.
 In cloning the gene for A-T (see Example 2), the strategy used was a
 standard strategy in identifying a disease gene with an -unknown protein
 product known as positional cloning, as is well known in the art. In
 positional cloning, the target gene is localized to a specific chromosomal
 region by establishing linkage between the disease and random genetic
 markers defined by. DNA polymorphisms. Definition of the smallest search
 interval for the gene by genetic analysis is followed by long-range
 genomic cloning and identification of transcribed sequences within the
 interval. The disease gene is then identified among these sequences,
 mainly by searching for mutations in patients.
 Several important and long sought disease genes were isolated recently in
 this way (Collins, 1992; Attree et al., 1992; Berger et al., 1992; Chelly
 et al., 1993; Vetrie et al., 1993; Trofatter et al., 1993; The
 Huntington's Disease Collaborative Research Group, 1993; The European
 Polycystic Kidney Disease Consortium, 1994; Miki et al., 1994).
 Two complementary methods were used for the identification of transcribed
 sequences (gene hunting): hybrid selection based on direct hybridization
 of genomic DNA with cDNAs from various sources (Parimoo et al., 1991;
 Lovett et al., 1991); and exon trapping (also called exon amplification),
 which identifies putative exons in genomic DNA by virtue of their splicing
 capacity (Church et al., 1993). In hybrid selection experiments, cosmid
 and YAC clones served to capture cross-hybridizing sequences in cDNA
 collections from placenta, thymus and fetal brain, using the magnetic bead
 capture protocol (Morgan et al., 1992; Tagle et al., 1993). In parallel
 experiments, YAC clones were bound to a solid matrix and used to select
 cDNA fragments from a heterogeneous cDNA collection representing several
 human tissues (Parimoo et al., 1993). The cosmids were also used for exon
 trapping with the pSPL3 vector (Church et al., 1994). The captured cDNA
 fragments and trapped exons were mapped back to the A-T region by
 hybridization to several radiation hybrids containing various portions of
 the 11q22-23 region (Richard et al., 1993; James et al., 1994), and to
 high-density grids containing all the YACs and cosmids spanning this
 interval. An extensive transcriptional map of the A-T region was thus
 constructed (Shiloh et al., 1994a).
 Pools of adjacent cDNA fragments and exons, expected to converge into the
 same transcriptional units, were used to screen cDNA libraries. A cluster
 of 5 cDNA fragments and 3 exons mapped in close proximity to the marker
 D11S535, where the location score for A-T had peaked (Lange et al., 1995).
 All these sequences hybridized to the same 5.9 kb of the cDNA clone, 7-9,
 (SEQ ID No:1) obtained from a fibroblast cDNA library.
 Hybridization of the 7-9 cDNA clone to the radiation hybrid panel indicated
 that the entire transcript was derived from the chromosome 11 locus. The
 full sequence of this clone (SEQ ID No:1) was obtained using a shotgun
 strategy, and found to contain 5921 bp which includes an open reading
 frame (ORF) of 5124 nucleotides, a 538 bp 3' untranslated region (3' UTR),
 and a 259 bp 5' non-coding sequence containing stop codons in all reading
 frames. (Genbank Accession No. U26455). Two Alu repetitive elements were
 observed at the 3' end of this clone and in nine smaller clones
 representing this gene from the same cDNA library. Since no
 polyadenylation signal was identified in these cDNA clones, their poly(A)
 tracts were assumed to be associated with the Alu element rather than
 being authentic poly(A) tails of these transcripts. This assumption was
 later supported when applicants identified a cDNA clone derived from the
 same gene in a leukocyte cDNA library, with an alternative 3' UTR
 containing a typical polyadenylation signal. Alignment of the cDNA with
 the genomic physical map showed that the corresponding gene is transcribed
 from centromere to telomere.
 Hybridization of a probe containing the entire ORF of clone 7-9 to northern
 blots from various tissues and cell lines revealed a major transcript of
 12 kb, later shown to be 13 kb, in all tissues and cell types examined,
 and minor species of various sizes in several tissues, possibly
 representing alternatively spliced transcripts of the corresponding gene
 or other homologous sequences. Genomic sequencing later identified the 5'
 non-coding region of clone 7-9 as sequences of the unspliced adjacent
 intron. Two other cDNA clones from a leukocyte cDNA library were found to
 contain this intronic sequence in their 5' ends. These clones may
 represent splicing intermediates.
 The 7-9 cDNA clone represents only part of the ATM gene transcript.
 Successive screening of randomly-primed cDNA libraries identified a series
 of partly overlapping cDNA clones and enabled the construction of a cDNA
 contig of about 10 Kb (FIG. 2). The gene coding for this transcript spans
 about 150 Kb of genomic DNA.
 The composite cDNA of 9860 bp (GenBank Accession No. U33841; SEQ ID No:2)
 includes an open reading frame of 9168 nucleotides, a 538 bp 3'
 untranslated region (UTR), and a 164 bp 5' UTR containing stop codons in
 all reading frames. The sequence surrounding the first in-frame initiation
 codon (ACCATGA) resembles the consensus sequence proposed by Kozak et al
 (1987) for optimal initiation of translation, (A/G)CCATGG. No
 polyadenylation signal was found at the 3' UTR. The same poly(A) tail was
 found in all cDNA clones and 3' RACE products isolated to date in
 applicant's laboratory, however, this poly(A) tail most likely belongs to
 the Alu element contained in the 3' UTR.
 Sequencing and PCR analysis of 32 partial ATM cDNA clones, obtained from 11
 cDNA libraries representing 8 different tissues, have been colinear over
 the coding region, except when they contained unspliced intronic
 sequences. Thus, alternative splicing within the ATM coding region may not
 occur, or may take place at a very low frequency, or be restricted to a
 cell type not yet explored.
 The invention further provides a purified protein (SEQ ID No:3) as encoded
 by the ATM gene and analogs and mutations thereof (SEQ ID No:2). The
 present invention further provides for mutations in SEQ ID No:3 which
 cause ataxia-telangiectasia, for example, as set forth in Tables 2 and 3.
 Th2 ATM Open Reading Frame (SEQ ID No:2) product is a large protein of 3056
 amino acids, with an expected molecular weight-of 350.6 kDa. The ATM gene
 product (SEQ ID No:3) contains a PI-3 kinase signature at codons
 2855-2875, and a potential leucine zipper at codons 1217-1238. The
 presence of this leucine zipper may suggest possible dimerization of the
 ATM protein or interaction with additional proteins. No nuclear
 localization signal, transmembrane domains or other motifs were observed
 in this protein sequence.
 The ATM gene product is a member of a family of large proteins that share a
 highly conserved carboxy-terminal region of about 300 amino acids showing
 high sequence homology to the catalytic domain of PI-3 kinases. Among
 these proteins are Tellp and Meclp in budding yeast, rad3p in fission
 yeast, the TOR proteins in yeast and their mammalian counterpart, FRAP
 (RAFT1), MEI-41 in Drosophila melanogaster, and the catalytic subunit of
 DNA-dependent protein kinase (DNA-PKcs) in mammals. All of these proteins
 are implicated in cell cycle control and some of them, like Mec1p, rad3p
 and DNA-PKcs are involved in response to DNA damage (Table 4). The central
 core of the PI-3 kinase-like domain contains two subdomains with highly
 conserved residues present in nearly all kinases, including protein and
 PI-3 kinases. The residues Asp and Asn (at positions 2870 and 2875 in
 ATM), and the triplet Asp-Phe-Gly (at positions 2889-2891), which
 represents the most highly conserved short stretch in the protein kinase
 catalytic domain, have been implicated in the binding of ATP and
 phosphotransferase activity. Mutations in the genes encoding these
 proteins result in a variety of phenotypes that share features with A-T,
 such as radiosensitivity, chromosomal instability, telomere shortening.,
 and defective cell cycle checkpoints (reviewed by Savitsky et al., 1995a
 and b; Zakian, 1995).
 A possible working model for the ATM protein's function is DNA-PK, a
 serine/threonine protein kinase that is activated in vitro by DNA
 double-strand breaks and responds by phosphorylating several regulator
 proteins (Gottlieb and Jackson, 1994). The ATM protein may be responsible
 for conveying a signal evoked by a specific DNA damage to various
 checkpoint systems, possibly via lipid or protein phosphorylation.
 The present invention further includes a recombinant protein encoded by SEQ
 ID No:2 or SEQ ID No:3 or analogs thereof. This recombinant protein is
 isolated and purified by techniques known to those skilled in the art.
 An analog will be generally at least 70% homologous over any portion that
 is functionally relevant. In more preferred embodiments, the homology will
 be at least 80% and can approach 95% homology to the ATM protein. The
 amino acid sequence of an analog may differ from that of the ATM protein
 when at least one residue is deleted, inserted or substituted but the
 protein remains functional and does not cause A-T. Differences in
 glycosylation can provide analogs.
 The present invention provides an antibody, either polyclonal or
 monoclonal, which specifically binds to epitopes on the
 polypeptide/protein encoded by the ATM gene, or mutant epitopes. In
 preparing the antibody, the protein (with and without mutations) encoded
 by the ATM gene and polymorphisms thereof is used as a source of the
 immunogen. Peptide amino acid sequences isolated from the amino acid
 sequence as set forth in SEQ ID No:3 or mutant peptide sequences can also
 be used as an immunogen.
 The present invention also provides antibodies against the following
 peptides:
 HEPANSSASQSTDLC (SEQ ID No:4),
 CKRNLSDIDQSFDKV (SEQ ID No:5),
 PEDETELHPTLNADDQEC (SEQ ID No:6), and
 CKSLASFIKKPFDRGEVESMEDDTNG (SEQ ID No:7).
 The antibodies may be either monoclonal or polyclonal. Conveniently, the
 antibodies may be prepared against a synthetic peptide based on the
 sequence, or prepared recombinantly by cloning techniques or the natural
 gene product and/or portions thereof may be isolated and used as the
 immunogen. Such proteins or peptides can be used to produce antibodies by
 standard antibody production technology well known to those skilled in the
 art as described generally in Harlow and Lane, Antibodies: A Laboratory
 Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
 1988.
 For producing polyclonal antibodies a host, such as a rabbit or goat, is
 immunized with the protein or peptide, generally with an adjuvant and, if
 necessary, coupled to a carrier; antibodies to the protein are collected
 from the sera.
 For producing monoclonal antibodies, the technique involves
 hyperimmunization of an appropriate donor, generally a mouse, with the
 protein or peptide fragment and isolation of splenic antibody producing
 cells. These cells are fused to a cell having immortality, such as a
 myeloma cell, to provide a fused cell hybrid which has immortality and
 secretes the required antibody. The cells are then cultured, in bulk, and
 the monoclonal antibodies harvested from the culture media for use.
 The antibody can be bound to a solid support substrate or conjugated with a
 detectable moiety or be both bound and conjugated as is well known in the
 art. (For a general discussion of conjugation of fluorescent or enzymatic
 moieties see Johnstone and Thorpe, Immunochemistry in Practice, Blackwell
 Scientific Publications, Oxford, 1982.) The binding of antibodies to a
 solid support substrate is also well known in the art. (see for a general
 discussion Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring
 Harbor Laboratory Publications, New York, 1988) The detectable moieties
 contemplated with the present invention can include, but are not limited
 to, fluorescent, metallic, enzymatic and radioactive markers such as
 biotin, gold,. ferritin, alkaline phosphatase, .beta.-galactosidase,
 peroxidase, urease, fluorescein, rhodamine, tritium, .sup.14 C and
 iodination.
 The present invention provides vectors comprising an expression control
 sequence operatively linked to the nucleic acid sequence of the ATM gene,
 SEQ ID No:2 and portions thereof as well as mutant sequences which lead to
 the expression of A-T. The present invention further provides-host cells,
 selected from suitable eucaryotic and procaryotic cells, which are
 transformed with these vectors.
 Using the present invention, it is possible to transform host cells,
 including E. coli, using the appropriate vectors so that they carry
 recombinant DNA sequences derived from the ATM transcript or containing
 the entire ATM transcript in its normal form or a mutated sequence
 containing point mutations, deletions, insertions, or rearrangements of
 DNA. Such transformed cells allow the study of the function and the
 regulation of the A-T gene. Use of recombinantly transformed host cells
 allows for the study of the mechanisms of A-T and, in particular it will
 allow for the study of gene function interrupted by the mutations in the
 A-T gene region.
 Vectors are known or can be constructed by those skilled in the art and
 should contain all expression elements necessary to achieve the desired
 transcription of the sequences. Other beneficial characteristics can also
 be contained within the vectors such as mechanisms for recovery of the
 nucleic acids in a different form. Phagemids are a specific example of
 such beneficial vectors because they can be used either as plasmids or as
 bacteriophage vectors. Examples of other vectors include viruses such as
 bacteriophages, baculoviruses and retroviruses, DNA viruses, cosmids,
 plasmids and-other recombination vectors. The vectors can also contain
 elements for use in either procaryotic or eucaryotic host systems. One of
 ordinary skill in the art will know which host systems are compatible with
 a particular vector.
 The vectors can be introduced into cells or tissues by any one of a variety
 of known methods within the art. Such methods can be found generally
 described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
 Springs Harbor Laboratory, New York (1992), in Ausubel et al., Current
 Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland
 (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich.
 (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor, Mich. (1995)
 and Gilboa et al (1986) and include, for example, stable or transient
 transfection, lipofection, electroporation and infection with recombinant
 viral vectors. Introduction of nucleic acids by infection offers several
 advantages over the other listed methods. Higher efficiency can be
 obtained due to their infectious nature. See also U.S. Pat. Nos. 5,487,992
 and 5,464,764. Moreover, viruses are very specialized and typically infect
 and propagate in specific cell types. Thus, their natural specificity can
 be used to target the vectors to specific cell types in vivo or within a
 tissue or mixed culture of cells. Viral vectors can also be modified with
 specific receptors or ligands to alter target specificity through receptor
 mediated events.
 Recombinant methods known in the art can also be used to achieve the sense,
 antisense or triplex inhibition of a target nucleic acid. For example,
 vectors containing antisense nucleic acids can be employed to express
 protein or antisense message to reduce the expression of the target
 nucleic acid and therefore its activity.
 A specific example of DNA viral vector for introducing and expressing
 antisense nucleic acids is the adenovirus derived vector Adenop53TK. This
 vector expresses a herpes virus thymidine kinase (TK) gene for either
 positive or. negative selection and an expression cassette for desired
 recombinant sequences such as antisense sequences. This vector can be used
 to infect cells that have an adenovirus receptor which includes most
 cancers of epithelial origin as well as others. This vector as well as
 others that exhibit similar desired functions can be used to treat a mixed
 population of cells include, for example, an in vitro or ex vivo culture
 of cells, a tissue or a human subject.
 Additional features can be added to the vector to ensure its safety and/or
 enhance its therapeutic efficacy. Such features include, for example,
 markers that can be used to negatively select against cells infected with
 the recombinant virus. An example of such a negative selection marker is
 the TK gene described above that confers sensitivity to the anti-viral
 gancyclovir. Negative selection is therefore a means by which infection
 can be controlled because it provides inducible suicide through the
 addition of antibiotic. Such protection ensures that if, for example,
 mutations arise that produce altered forms of the viral vector or
 sequence, cellular transformation will not occur. Features that limit
 expression to particular cell types can also be included. Such features
 include, for example, promoter and regulatory elements that are specific
 for the desired cell type.
 Recombinant viral vectors are another example of vectors useful for in vivo
 expression of a desired nucleic acid because they offer advantages such as
 lateral infection and targeting specificity. Lateral infection is inherent
 in the life cycle of, for example, retrovirus and is the process by which
 a single infected cell produces many progeny virions that bud off and
 infect neighboring cells. The result is that a large area becomes rapidly
 infected, most of which was not initially infected by the original viral
 particles. This is in contrast to vertical-type of infection in which the
 infectious agent spreads only through daughter progeny. Viral vectors can
 also be produced that are unable to spread laterally. This characteristic
 can be useful if the desired purpose is to introduce a specified gene into
 only a localized number of targeted cells.
 As described above, viruses are very specialized infectious agents that
 have evolved, in many cases, to elude host defense mechanisms. Typically,
 viruses infect and propagate in specific cell types. The targeting
 specificity of viral vectors utilizes its natural specificity to
 specifically target predetermined cell types and thereby introduce a
 recombinant gene into the infected cell. The vector to be used in the
 methods of the invention will depend on desired cell type to be targeted.
 For example, if breast cancer is to be treated, then a vector specific for
 such epithelial cells should be used. Likewise, if diseases or
 pathological conditions of the hematopoietic system are to be treated,
 then a viral vector that is specific for blood cells and their precursors,
 preferably for the specific type of hematopoietic cell, should be used.
 Retroviral vectors can be constructed to function either as infectious
 particles or to undergo only a single initial round of infection in the
 former case, the genome of the virus is modified so that it maintains all
 the necessary genes, regulatory sequences and packaging signals to
 synthesize new viral proteins and RNA. Once these molecules are
 synthesized, the host cell packages the RNA into new viral particles which
 are capable of undergoing further rounds of infection. The vector's genome
 is also engineered to encode and express the desired recombinant gene. In
 the case of non-infectious viral vectors, the vector genome is usually
 mutated to destroy the viral packaging signal that is required to
 encapsulate the RNA into viral particles. Without such a signal, any
 particles that are formed will not contain a genome and therefore cannot
 proceed through subsequent rounds of infection. The specific type of
 vector will depend upon the intended application. The actual vectors are
 also known and readily available within the art or can be constructed by
 one skilled in the art using well-known methodology.
 If viral vectors are used, for example, the procedure can take advantage of
 their target specificity and consequently, do not have to be administered
 locally at the diseased site. However, local administration may provide a
 quicker and more effective treatment, administration can also be performed
 by, for example, intravenous or subcutaneous injection into the subject.
 Injection of the viral vectors into a spinal fluid can also be used as a
 mode of administration, especially in the case of neurodegenerative
 diseases. Following injection, the viral vectors will circulate until they
 recognize host cells with the appropriate target specificity for
 infection.
 Transfection vehicles such as liposomes can also be used to introduce the
 non-viral vectors described above into recipient cells within the
 inoculated area. Such transfection vehicles are known by one skilled
 within the art.
 The present invention includes the construction of transgenic and knockout
 organisms that exhibit the phenotypic manifestations of A-T. The present
 invention provides for transgenic ATM gene and mutant ATM gene animal and
 cellular (cell lines) models as well as for knockout ATM models. The
 transgenic model can include those carrying the sequence set forth SEQ ID
 Nos:2,8,9 (or 10). These models are constructed using standard methods
 known in the art and as set forth in U.S. Pat. Nos. 5,487,992, 5,464,764,
 5,387,742, 5,360,735, 5,347,075, 5,298,422, 5,288,846, 5,221,778,
 5,175,385, 5,175,384,5,175,383, 4,736,866 as well as Burke and Olson,
 (1991), Capecchi, (1989), Davies et al., (1992), Dickinson et al., (1993),
 Huxley et al., (1991), Jakobovits et al., (1993), Lamb et al., (1993),
 Rothstein, (1991), Schedl et al., (1993), Strauss et al., (1993). Further,
 patent applications WO 94/23049, WO 93/14200, WO 94/06908, WO 94/28123
 also provide information. See also in general Hogan et al "Manipulating
 the Mouse Embryo" Cold Spring Harbor Laboratory Press, 2nd Edition (1994).
 According to the present invention, there is provided a method for
 diagnosing and detecting carriers of the defective gene responsible for
 causing A-T (see Example 4).
 The present invention further provides methods for detecting normal copies
 of the ATM gene and its gene product. Carrier detection is especially
 important since A-T mutations underlie certain cases of cancer
 predisposition in the general population. Identifying the carriers-either
 by their defective gene or by their missing or defective protein(s)
 encoded thereby, leads to earlier and more consistent diagnosis of A-T
 gene carriers. Thus, since carriers of the disease are more likely to be
 cancer-prone and/or sensitive to therapeutic applications of radiation,
 better surveillance and treatment protocols can be initiated for them.
 Conversely, exclusion of A-T heterozygotes from patients undergoing
 radiotherapy can allow for establishing routinely higher dose schedules
 for other cancer patients thereby improving the efficacy of their
 treatment.
 Briefly, the methods comprise the steps of obtaining a sample from a test
 subject, isolating the appropriate test material from the sample and
 assaying for the target nucleic acid sequence or gene product. The sample
 can be tissue or bodily fluids from which genetic material and/or proteins
 are isolated using methods standard in the art. For example, DNA can be
 isolated from lymphocytes, cells in amniotic fluid and chorionic villi
 (Llerena et al., 1989).
 More specifically, the method of carrier detection is carried out by first
 obtaining a sample of either cells or bodily fluid from a subject.
 Convenient methods for obtaining a cellular sample can include collection
 of either mouth wash fluids or hair roots. A cell sample could be amniotic
 or placental cells or tissue in the case of a prenatal diagnosis. A crude
 DNA could be made from the cells (or alternatively proteins isolated) by
 techniques well known in the art. This isolated target DNA is then used
 for PCR analysis (or alternatively, Western blot analysis for proteins
 from a cell line established from the subject) with appropriate primers
 derived from the gene sequence by techniques well known in the art. The
 PCR product would then be tested for the presence of appropriate sequence
 variations in order to assess genotypic A-T status of the subject.
 The specimen can be assayed for polypeptides/proteins by
 immunohistochemical and immunocytochemical staining (see generally Stites
 and Terr, Basic and Clinical Immunology, Appleton and Lange, 1994), ELISA,
 RIA, immunoblots, Western blotting, immunoprecipitation, functional assays
 and protein truncation test. In preferred embodiments, Western blotting,
 functional assays and protein truncation test (Hogervorst et al., 1995)
 will be used. mRNA complementary to the target nucleic acid sequence can
 be assayed by in situ hybridization, Northern blotting and reverse
 transcriptase-polymerase chain reaction. Nucleic acid sequences can be
 identified by in situ hybridization, Southern blotting, single strand
 conformational polymorphism, PCR amplification and DNA-chip analysis using
 specific primers. (Kawasaki, 1990; Sambrook, 1992; Lichter et al, 1990;
 Orita et al, 1989; Fodor et al., 1993; Pease et al., 1994)
 ELISA assays are well known to those skilled in the art. Both polyclonal
 and monoclonal antibodies can be used in the assays. Where appropriate
 other immunoassays, such as radioimmunoassays (RIA) can be used as are
 known to those in the art. Available immunoassays are extensively
 described in the patent and scientific literature. See, for example, U.S.
 Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987;
 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;
 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521 as well as
 Sambrook et al, 1992.
 Current mutation data (as shown in Tables 2 and 3) indicate that A-T is a
 disease characterized by considerable allelic heterogenicity. It is not
 surprising that there are hundreds (or even thousands) of ATM mutations
 (as is the case for cystic fibrosis and BRCA1) as shown in Table 3. Thus,
 it will be important for a successful mutation screen to be able to detect
 all possible nucleotide alterations in the ATM gene, rather than being
 focused on a limited subset (see Example 4). Methods including direct
 sequencing of PCR amplified DNA or RNA or DNA chip hybridization (Fodor et
 al., 1993; Pease et al., 1994) can be applied along with other suitable
 methods known to those skilled in the art.
 In order to use the method of the present invention for diagnostic
 applications, it is advantageous to include a mechanism for identifying
 the presence or absence of target polynucleotide sequence (or
 alternatively proteins). In many hybridization based diagnostic or
 experimental procedures, a label or tag is used to detect or visualize for
 the presence or absence of a particular polynucleotide sequence.
 Typically, oligomer probes are labelled with radioisotopes such as .sup.32
 P or .sup.35 S (Sambrook, 1992) which can be detected by methods well
 known in the art such as autoradiography. Oligomer probes can also be
 labelled by non-radioactive methods such as chemiluminescent materials
 which can be detected by autoradiography (Sambrook, 1992). Also,
 enzyme-substrate based labelling and detection methods can be used.
 Labelling can be accomplished by mechanisms well known in the art such as
 end labelling (Sambrook, 1992), chemical labelling, or by hybridization
 with another labelled oligonucleotide. These methods of labelling and
 detection are provided merely as examples and are not meant to provide a
 complete and exhaustive list of all the methods known in the art.
 The introduction of a label for detection purposes can be accomplished by
 attaching the label to the probe prior to hybridization.
 An alternative method for practicing the method of the present invention
 includes the step of binding the target DNA to a solid support prior to
 the application of the probe. The solid support can be any material
 capable of binding the target DNA, such as beads or a membranous material
 such as nitrocellulose or nylon. After the target DNA is bound to the
 solid support, the probe oligomers is applied.
 Functional assays can be used for detection of A-T carriers-or affected
 individuals. For example, if the ATM protein product is shown to have PI
 3-kinase or a protein kinase biochemical activity which can be assayed in
 an accessible biological material, such as serum, peripheral leukocytes,
 etc., then homozygous normal individuals would have approximately normal
 biological activity and serve as the positive control. A-T carriers would
 have substantially less than normal biological activity, and affected
 (i.e. homozygous) individuals would have even less biological activity and
 serve as a negative control. Such a biochemical assay currently serves as
 the basis for Tay-Sachs carrier detection.
 The present invention provides a method for a rapid and efficient method to
 identify any mutations in small amounts of RNA, such as those that are
 obtained from as little as 100 .mu.l of peripheral blood. RNA is extracted
 from the sample, using the Tri-Reagent system (Molecular Research Center,
 Cincinnati Ohio) or other equivalent method and it is subjected to reverse
 transcription as described by Gilad et al (1996). A selected open reading
 frame is then amplified using RT-PCR as described herein below, and the
 resulting products are further amplified using nested PCR primers. The
 products of these reactions are then subjected to restriction endonuclease
 fingerprinting (REF) as described hereinbelow.
 This method uses the cDNA obtained from the RNA for only one PCR, the
 products of which later serve for further analysis. Therefore, only
 minimal amounts of RNA need be extracted, reducing costs.
 Using this method RNA isextracted from a sample and subjected to-reverse
 transcription (Gilad et al., 1996). The entire ATM open reading frame (SEQ
 ID No:2) is then amplified using RT-PCR as described herein below, and the
 resulting products are further amplified using nested PCR primers (SEQ ID
 Nos:82-91; see herein below). The products of these reactions are then
 subjected to restriction endonuclease fingerprinting (REF) as described
 hereinbelow.
 However, when only genomic DNA (as for example old tumor specimens) is
 available the preferred embodiment to undertake mutation analysis requires
 individual amplification of exons or groups of exons. To undertake this
 analysis primer pairs have been developed based on the information set
 forth in Table 1 and in Table 5 which is a further amplification of Table
 1. Primers are selected from the flanking sequences using standard
 computer algorithms as are known in the art. Shown below are five examples
 of primer pairs which can be used for the analysis.
 Primers Exons 4 and 5
 5' primer: 5'-cacacctctttctctctatatatg-3' (SEQ ID No:82)
 3' primer: 5'-cacacaaaagtaatatcacaacag-3' (SEQ ID No:83)
 Primers Exon 17
 5' primer: 5'-gtatgtccaagatcaaagtacac-3' (SEQ ID No:84)
 3' primer: 5'-ggtgacagagaaagatcctatctc-3' (SEQ ID No:85)
 Primers Exon 25
 5' primer: 5'-ctggaatatgctttggaaagtagg-3' (SEQ ID No:86)
 3' primer: 5'-ccaaacttggtgaagtaatttatgg-3' (SEQ ID No:87)
 Primers Exon 34
 5' primer: 5'-cacaggcttaaccaatacgtg-3' (SEQ ID No:88)
 3' primer: 5'-caggtagaaatagcccatgtc-3' (SEQ ID No:89)
 Primers Exon 46
 5' primer: 5'-gtcctttggtgaagctatttatac-3' (SEQ ID No:90)
 3' primer: 5'-ccaagtctttatctcttcatcaatgc-3' (SEQ ID No:91)
 The present invention also provides a kit for diagnosis and detection of
 the defective A-T gene in a population. In general the population will be
 one that has been characterized for ATM mutations. For example Moroccan
 Jews in which only one mutation is known, or a population such as the
 Amish in which also one mutation is known. Each kit would be customized to
 the population being screened. The kit includes a molecular probe(s)
 complementary to genetic sequences of the defective gene which causes
 ataxia-telangiectasia (A-T) in that population and suitable labels for
 detecting hybridization of the molecular probe and the defective gene
 thereby indicating the presence of the defective gene. The molecular probe
 has a DNA sequence complementary to mutant sequences. Alternatively, the
 kit can contain reagents and antibodies for detection of mutant proteins.
 The above discussion provides a factual basis for the use and
 identification of the ataxia-telangiectasia gene and gene products and
 identification of carriers as well as construction of transgenic
 organisms. The methods used in the present invention can be shown by the
 following non-limiting example and accompanying figures.
 EXAMPLES
 Materials and Methods
 General methods in molecular biology: Standard molecular biology techniques
 known in the art and not specifically described were generally followed as
 in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs
 Harbor Laboratory, New York (1989, 1992), and in Ausubel et al., Current
 Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.
 (1989). Protein analysis techniques are as generally described in Coligan
 et al., Current Protocols in Immunology, John Wiley and Sons, Baltimore,
 Md. (1992, 1994).
 RT-PCR
 Following reverse transcription (Gilad et al., 1996), RT-PCR is performed
 in 25 .mu.l reaction mix. One tenth (1 .mu.l ) of the reverse
 transcription products are added to the reaction mix. The reaction mix
 contains 2U of Expand Long Template PCR System (Boehringer Mannheim,
 Mannheim, Germany), in an appropriate buffer containing 1.75 mM
 MgCl.sub.2, 1.5 .mu.g of Antitaq mAb (Chimerx), 0.2 mM dNTPs (Pharmacia)
 and 1 mM of each of the following primers:
 a. 5'-gtgcagtgaggcatacatcac-3' (SEQ ID No:64)
 b. 5'-aaggctgaatgaaagggtaattc-3' (SEQ ID No:65)
 PCR Conditions
 After denaturation for 3 minutes at 94.degree. C., 5 cycles of
 amplification are performed as follows: 20 seconds at 93.degree. C., 1
 minute at 64.degree. C. and 8 minutes at 68.degree. C., followed by 30
 cycles of 20 seconds at 93.degree. C., 1 minute at 60.degree. C. and 7.5
 minutes at 68.degree. C. Final extension is then performed for 10 minutes
 at 68.degree. C.
 Restriction Endonuclease Fingerprinting (REF)
 The open reading frame of the ATM transcript was divided into eight partly
 overlapping PCR fragments. Each fragment spans 1.0-1.6 kb and is defined
 by two PCR primers as follows.
 REF1.
 a. 5'-caccctgctgcccagatatg-3' (SEQ ID No:66)
 b. 5'-cttataccacgaaaggtaatacac-3' (SEQ ID No:67)
 REF2.
 a. 5'-gaggtcaaacctagaaagctcac-3' (SEQ ID No:68)
 b. 5'-cctctcctttgttagatgcc-3' (SEQ ID No:69)
 REF3.
 a. 5'-ctaggtcaaagcaatatggactc-3' (SEQ ID No:70)
 b. 5'-catgcgatggaaaatgaggtg-3' (SEQ ID No:71)
 REF4.
 a. 5'-cagagattgtggtggagttattg-3' (SEQ ID No:72)
 b. 5'-gcattatgaaggtccactgaag-3' (SEQ ID No:73)
 REF5.
 a. 5'-cttcagtggaccttcataatgc-3' (SEQ ID No:74)
 b. 5'-ccatacaaactatctggctcc-3' (SEQ ID No:75)
 REF6.
 a. 5'-ctggaataagtttacaggatcttc-3' (SEQ ID No:76)
 b. 5'-gatgatttcatgtagttttcaattc-3' (SEQ ID N6:77)
 REF7.
 a. 5'-gatggagaaagtagtgatgagc-3' (SEQ ID No:78)
 b. 5'-agtcaccagatttccatattctc-3' (SEQ ID No:79)
 REF8.
 a. 5 '-aagatgttgttgtccctactatg-3' (SEQ ID No:80)
 b. 5'-aaggctgaatgaaagggtaattc-3' (SEQ ID No:81)
 Patient and family resources: A cell line repository was established
 containing 230 patient cell lines and 143 cell lines from healthy members
 of Moroccan Jewish, Palestinian Arab and Druze families. Some of these
 pedigrees are highly inbred and unusually large (Ziv et al., 1991; Ziv,
 1992). In view of the large number of meiotic events required for
 high-resolution linkage analysis, applicants collaborated with Dr. Carmel
 McConville (University of Birmingham, UK) and Dr. Richard Gatti (UCLA, Los
 Angeles, Calif.), who have also established extensive repositories of A-T
 families. Linkage analysis was conducted on a pool of 176 families.
 Example 1
 Definition of the A-T interval by genetic analysis: Studies based only on
 analysis of Israeli A-T families enabled localization of the A-T(C) gene
 at 11q22-23 (Ziv, 1991), and confirmed the localization of A-T(A) mutation
 in Palestinians to the same region (Ziv et al., 1992). Studies with the
 Birmingham group further narrowed the major A-T interval to 4
 centimorgans, between D11S611 and D11S1897 (McConville et al., 1993), and
 subsequently to 3 centimorgans, between GRIA4 and D11S1897 (Ambrose et
 al., 1994a; McConville et al., 1994) (see also Shiloh, 1995, and FIG. 1).
 All these studies were conducted with biallelic markers, whose power is
 limited by their low polymorphic information content (PIC). The recently
 discovered microsatellite markers based on variable numbers of tandem
 simple repeats (Litt-and Luty, 1989; Weber and May, 1989) are much more
 powerful due to their high degree of polymorphism. Microsatellite markers
 were used to saturate the A-T region using two approaches. The first, was
 based on physical mapping of microsatellite markers generated by others
 which were loosely linked to chromosome 11q.
 Mapping experiments were conducted using YAC and cosmid contigs which
 allowed precise, high-resolution localization of DNA sequences in this
 region of chromosome 11. These experiments led to the localization of 12
 microsatellites at the A-T region (Vanagaite et al., 1994a; Vanagaite et
 al., 1995).
 The second approach was based on generating new microsatellites within the
 YAC contig. A rapid method for the identification of polymorphic
 CA-repeats in YAC clones was set up (Rotman, 1995) resulting in the
 generation of twelve new markers within the A-T locus (Vanagaite et al.,
 1995; Rotman et al., 1995; Rotman et al., 1994b). Hence, the high-density
 microsatellite map constructed in this manner contained a total of 24 new
 microsatellite markers and spans the A-T locus and flanking sequences,
 over a total of six megabases (Vanagaite et al., 1995).
 Repeated linkage analysis on the entire cohort of A-T families indicated
 that the A-T(A) locus was definitely located within a 1.5 megabase region
 between DlS1819 and D11S1818 (Gatti et al., 1994) as shown in FIG. 1 and
 in Shiloh (1995), with a clear peak of the cumulative lod score under
 D11S535 (Lange et al., 1994).
 Concomitant with these studies, linkage disequilibrium (LD) analysis of
 Moroccan-Jewish A-T patients was conducted. LD refers to the non-random
 association between alleles at two or more polymorphic loci (Chakravarti
 et al., 1984). LD between disease loci and linked markers is a useful tool
 for the fine localization of disease genes (Chakravarti et al., 1984;
 Kerem et al. 1989; Ozelius et al., 1992; Sirgo et al.:, 1992;. Hastbacka
 et al., 1992; Mitchison et al., 1993). LD is particularly powerful in
 isolated ethnic groups, where the number of different mutations at a
 disease locus is likely to be low (Hastbacka et al., 1992; Lehesjoki et
 al., 1993; Aksentijevitch et al., 1993). Early on, applicants observed
 very significant LD (p&lt;0.02-p&lt;0.001) between A-T and markers along the
 D11S1817-D11S927 region in the patients of the sixteen Moroccan-Jewish A-T
 families identified in Israel (Oskato et al., 1993). Further analysis with
 the new markers narrowed the peak of linkage disequilibrium to the
 D11S384-D11S1818 region as shown in FIG. 1.
 Haplotype analysis indicated that all of the mutant chromosomes carry the
 same D11S384-D11S1818 haplotype, suggesting a founder effect for A-T in
 this community, with one mutation predominating.
 Example 2
 Sequencing the ATM Gene
 Cloning the disease locus in a contig (set of overlapping clones) was
 essential in isolating the A-T disease gene. The entire A-T locus and
 flanking region in a contig of yeast artificial chromosomes (YACs) was
 cloned by methods well known in the art (Rotman et al. 1994c; Rotman et
 al., 1994d). This contig was instrumental in the construction of the
 microsatellite map of the region (Vanagaite et al., 1995) and subsequently
 enabled construction of cosmid contigs extending over most of the interval
 D11S384-D11S1818. Cosmids corresponding to the YAC clones were identified
 in a chromosome 11-specific cosmid library supplied by Dr. L. Deaven (Los
 Alamos National Laboratory) and were ordered into contigs by identifying
 overlaps as shown in FIG. 1.
 Isolation of the A-T gene: Transcribed sequences were systematically
 identified based on two complementary methods:
 1. Use of an improved direct selection method based on magnetic bead
 capture (MBC) of cDNAs corresponding to genomic clones (Morgan et al.,
 1992; Tagle et al., 1993). In several, large-scale experiments YAC or
 cosmid DNA was biotinylated and hybridized to PCR-amplified cDNA from
 thymus, brain and placenta. Genomic DNA-cDNA complexes were captured using
 streptavidin-coated magnetic beads which was followed with subsequent
 elution, amplification, and cloning of captured cDNAs. The cDNA inserts
 were excised from a gel, self-ligated to form concatamers and sonicated to
 obtain random fragments. These fragments were size fractionated by gel
 electrophoresis, and the 1.0-1.5 Kb fraction-was extracted from the gel
 and subcloned in a plasmid vector. The end portions of individual clones
 were sequenced using vector-specific primers, in an automated sequencer
 (Model 373A, Applied Biosystems), and the sequences were aligned using the
 AutoAssembler program (Applied Biosystems Division, Perkin-Elmer
 Corporation). In the final sequence each nucleotide position represents at
 least 3 independent overlapping readings.
 YACs were also used and were no less efficient than cosmids as starting
 material for MBC, with more than 50% of the products mapping back to the
 genomic clones. However, when a small panel of radiation hybrids spanning
 the A-T region was used to test the cDNA fragments, it was found that some
 clones that hybridized back to the YACs and cosmids were not derived from
 this region. This pitfall probably stems from limited homology between
 certain portions of different genes, and points up the necessity to use
 radiation hybrid mapping when testing the authenticity of the captured
 sequences, and not to rely solely on cloned DNA for this purpose.
 Homology searches in sequence databases showed that only one of the first
 105 cDNA fragments mapped to the A-T region was homologous to a sequence
 previously deposited in one of the databases, as an expressed sequence tag
 (EST).
 2. Exon amplification, also termed "exon trapping" (Duyk et al., 1990;
 Buckler et al., 1991), is based on cloning genomic fragments into a vector
 in which exon splice sites are flagged by splicing to their counterpart
 sites in the vector. This method of gene identification was expected to
 complement the MBC strategy, since it does not depend on the constitution
 of cDNA libraries or on the relative abundance of transcripts, and is not
 affected by the presence of repetitive sequences in the genomic clones. An
 improved version of this system (Church et al., 1993) that eliminated
 problems identified in an earlier version, including a high percentage of
 false positives and the effect of cryptic splice sites was utilized. Each
 experiment ran a pool of three to five cosmids with an average of two to
 five exons identified per cosmid. A total of forty five exons were
 identified.
 Sequence analysis and physical mapping indicated that MBC and exon
 amplification were complementary in identifying transcribed sequences.
 The availability of a deep cosmid contig enabled rapid and precise physical
 localization of the cDNA fragments and captured exons, leading to a
 detailed transcriptional map of the A-T region.
 Both MBC and exon amplification yielded short (100-1000 bp) transcribed
 sequences. Those sequences were used as anchor points in isolating
 full-length clones from twenty eight cDNA libraries currently at
 applicants disposal and which represented a variety of tissues and cell
 lines.
 Initial screening of the cDNA libraries by polymerase chain reaction (PCR)
 using primer sets derived from individual cDNA fragments or exons aided in
 the identification of the libraries most likely to yield corresponding
 cDNA clones.
 Large scale screening experiments were carried out in which most of the
 cDNA fragments and exons were used in large pools. In addition to the mass
 screening by hybridization, PCR-based screening methods and RACE (rapid
 amplification of cDNA ends) (Frohman et al., 1988; Frohman et al., 1994)
 was employed to identify full-length cDNAs.
 The above experiments resulted in the initial, identification and isolation
 of a cDNA clone designated 7-9 (Savitsky et al, 1995a), the complete
 sequence of which is set forth in SEQ ID No:1 and which is derived from a
 gene located under the peak of cumulative location score obtained by
 linkage analysis as shown in FIG. 1. The gene extends over some 300
 kilobases (kb) of genomic DNA and codes for two major mRNA species of 12
 kb and 10.5 kb in length. The 7-9 clone is 5.9 kb in length and,
 therefore, is not a full length clone.
 An open reading frame of 5124 bp within this cDNA encodes a protein with
 signature motifs typical of a group of signal transduction proteins known
 as phosphatidylinositol 3-kinases (PI 3-kinases). PI 3-kinases take part
 in the complex system responsible for transmitting signals from the outer
 environment of a cell into the cell. It is not clear yet whether the
 protein product of the corresponding gene encodes a lipid kinase or a
 protein kinase.
 The gene encoding the 7-9 cDNA clone was considered a strong A-T candidate
 and mutations were sought in patients. Southern blotting analysis revealed
 a homozygous deletion in this gene in affected members of Family N., an
 extended Palestinian Arab A-T family which has not been assigned to a
 specific complementation group. All the patients in this family are
 expected to be homozygous by descent for a single A-T mutation. The
 deletion includes almost the entire genomic region spanned by transcript
 7-9, and was found to segregate in the family together with the disease.
 This finding led to a systematic search for mutations in the 7-9
 transcript in additional patients, especially those previously assigned to
 specific complementation groups.
 The restriction endonuclease fingerprinting (REF) method (Liu and Sommer
 1995) was applied to reverse-transcribed and PCR-amplified RNA (RT-PCR)
 from A-T cell lines. Observation of abnormal REF patterns was followed by
 direct sequencing of the relevant portion of the transcript and repeated
 analysis of another independent RT product. In compound heterozygotes, the
 two alleles were separated by subcloning of RT-PCR products and
 individually sequenced. Genomic sequencing was conducted in some cases to
 confirm the sequence alteration at the genomic level. Additional family
 members were studied when available.
 Initially, ten sequence alterations (Table 2) were identified in the 7-9
 transcript in 13 A-T patients including two sibling pairs. Most of these
 sequence changes are expected to lead to premature truncation of the
 protein product, while the rest are expected to create in-frame deletions
 of 1-3 amino acid residues in this protein. While the consequences of the
 in-frame deletions remain to be investigated, it is reasonable to assume
 that they result in impairment of protein function. In one patient, AT3NG,
 the loss of a serine residue at position 1512 occurs within the PI3-kinase
 signature sequence. This well conserved domain is distantly related to the
 catalytic site of protein kinases, hence this mutation is likely to
 functionally affect the 7-9 protein.
 In view of the strong evidence that mutations in this gene are responsible
 for A-T, it was designated ATM (A-T, Mutated). Since these patients
 represent all complementation groups of the disease and considerable
 ethnic variability, these results indicate that the ATM gene alone is
 responsible for all A-T cases.
 In order to complete the cloning of the entire ATM open reading frame,
 fetal brain and colon random-primed libraries obtained from Stratagene
 (San Diego, Calif.) and an endothelial cell random-primed library (a gift
 of Dr. David Ginsburg, University of Michigan) were screened. A total of
 1.times.10.sup.6 pfu were screened at a density of 40,000 pfu per 140 mm
 plate, and replicas were made on Qiabrane filters (Qiagen), as recommended
 by the manufacturer. Filters were prehybridized in a solution containing
 6.times.SSC, 5.times.Denhardt's, 1% N-laurylsarcosyl, 10% dextran sulfate
 and 100 .mu.g/ml salmon sperm DNA for 2 hours at 65.degree. C.
 Hybridization was performed for 16 hrs under the same conditions with
 1.times.10.sup.6 cpm/ml of .sup.32 P-labelled probe, followed by final
 washes of 30 minutes in 0.25.times.SSC, 0.1% SDS at 60.degree. C. Positive
 clones were plaque-purified using standard techniques and sequenced. DNA
 sequencing was performed using an automated DNA sequencer (Applied
 Biosystems, model 373A), and the sequence was assembled using the
 AutoAssembler program (Applied Biosystems Division, Perkin-Elmer
 Corporation). In the final sequence, each nucleotide represents at least
 four independent readings in both directions.
 Database searches for sequence similarities were performed using the BLAST
 network service. Alignment of protein sequences and pairwise comparisons
 were done using the MACAW program, and the PILEUP and BESTFIT programs in
 the sequence analysis software package developed by the Genetics Computer
 Group at the University of Wisconsin.
 Example 3
 Detection of Mutations
 Determination of mutations: The recently discovered ATM gene is probably
 involved in a novel signal transduction system that links DNA damage
 surveillance to cell cycle control. A-T mutations affect a variety of
 tissues and lead to cancer predisposition. This striking phenotype
 together with the existence of "partial A-T phenotypes" endow the study of
 ATM mutations with special significance.
 Materials and Methods
 RT-PCR: Total RNA was extracted from cultured fibroblast or lymphoblast
 cells using the Tri-Reagent system (Molecular research Center, Cincinnati,
 Ohio). Reverse transcription was performed on 2.5 ug of total RNA in a
 final volume of 10 ul, using the Superscript II Reverse Transcriptase
 (Gibco BRL, Gaithersburg, Md.) in the buffer recommended by the supplier,
 and in the presence of 125 U/ml of RNAsin. (Promega) and 1 mM dNTPs
 (Pharmacia). Primers were either oligo(dT) (Pharmacia) or a specifically
 designed primer. The reaction products were used as templates for PCR
 performed with specific primers. These reactions were carried out in 50
 .mu.l containing 2 units of Taq DNA Polymerase (Boehringer Mannheim,
 Mannheim, Germany), 200 .mu.m dNTPs, 0.5 .mu.M of each primer, and one
 tenth of the RT-PCR products. The products were purified using the
 QIA-quick spin system (Qiagen, Hilden, Germany).
 Restriction endonuclease fingerprinting: The protocol of Liu and Sommer
 (1995) was followed with slight modifications. RT-PCR was performed as
 described above, using primers defining PCR products of 1.0-1.6 kb. One
 hundred ng of amplified DNA was digested separately with 5 or 6
 restriction endonucleases in the presence of 0.2 units of shrimp alkaline
 phosphatase (United States Biochemicals, Cleveland, Ohio). Following heat
 inactivation at 65.degree. C. for 10 minutes, the digestion products
 corresponding to the same PCR product were pooled, denatured at 96.degree.
 C. for 5 minutes and immediately chilled on ice. Ten ng of this fragment
 mixture was labeled in the presence of 6 .mu.Ci of [.gamma.-.sup.33 P]ATP
 and 1 unit of T4 polynucleotide kinase (New England Biolabs, Beverly,
 Mass.) at 37.degree. C. for 45 minutes. Twenty .mu.l of stop solution
 containing 95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene
 cyanol, and 10 mM NaOH were added, and the samples were boiled for 3
 minutes and quick-chilled on ice. Electrophoresis was performed in 5.6%
 polyacrylamide gels in 50 mM Tris-borate, pH 8.3, 1 mM EDTA at constant
 power of 12 W for 3 hours at room temperature, with a fan directed to the
 glass plates, keeping them at 22-24.degree. C. The gels were dried and
 subjected to autoradiography.
 Direct sequencing of PCR products: Five hundred ng of PCR products was
 dried under vacuum, resuspended in reaction buffer containing the
 sequencing primer, and the mixture was boiled and snap-frozen in liquid
 nitrogen. The Sequenase II system (Unites States Biochemicals) was used to
 carry out the sequencing reaction in the presence of 0.5 .mu.g of
 single-strand binding protein (T4 gene 3.2 protein, United States
 Biochemicals). The reaction products were treated with 0.1 .mu.g of
 proteinase K at 65.degree. C. for 15 minutes, separated on a 6%
 polyacrylamide gel, and visualized by autoradiography.
 Using the methods described herein above the ATM transcript was scanned for
 mutations in fibroblast and lymphoblast cell lines derived from an
 extended series of A-T patients from 13 countries, all of whom were
 characterized by the classical A-T phenotype. The analysis was based on
 RT-PCR followed by restriction endonuclease fingerprinting (REF). REF is a
 modification of the single-strand conformation polymoprphism (SSCP)
 method, and enables efficient detection of sequence alterations in DNA
 fragments up to 2 kb in length (Liu and Sommer, 1995).
 Briefly, after PCR amplification of the target region, multiple restriction
 endonuclease digestions are performed prior to SSCP analysis, in order to
 increase the sensitivity of the method and enable precise localization of
 a sequence alteration within the analyzed fragment. The coding sequence of
 the ATM transcript, which spans 9168 nucleotides (SEQ ID No:2) (Savitsky
 et al., 1995b), was thus divided into 8 partly overlapping portions of
 1.0-1.6 Kb, and each one was analyzed separately (see Example 4 also).
 Sequence alterations causing abnormal REF patterns were located and
 disclosed by direct sequencing. Mutations identified in this way were
 reconfirmed by repeating the RT-PCR and sequencing, or by testing the
 presence of the same mutations in genomic DNA.
 In compound heterozygotes, the two alleles were separated by subcloning and
 individually sequenced. In some cases, agarose gel electrophoresis showed
 large deletions in the ATM transcript manifested as RT-PCR products of
 reduced sizes. The breakpoints of such deletions were delineated by direct
 sequencing of these products.
 The 54 mutations identified to date in our patient cohort (Table 3, FIG. 4)
 include 44 new ones and 10 previously identified ones (Table 2).
 (Mutations in Table 3 are presehted according to the nomenclature proposed
 by Beaudet & Tsui (1993); nucleotide numbers refer to their positions in
 the sequence of the ATM transcript (accession number U33841); the first
 nucleotide of the open reading frame was designated +1.) These mutations
 were found amongst 65 A-T families: many are unique to a single family,
 while others are shared by several families, most notably the 4 nt
 deletion, 7517del4, which is common to 6 A-T families from South-Central
 Italy (Table 3). According to this sample, there is a considerable
 heterogeneity of mutations in A-T, and most of them are "private". The
 proportion of homozygotes in this sample is relatively high due to a high
 degree of consanguinity the populations studied. It should be noted,
 however, that apparently homozygous patients from non-consanguineous
 families may in fact be compound heterozygotes with one allele not
 expressed.
 This series of 54 A-T mutations is dominated by deletions and insertions.
 The smaller ones, of less than 12 nt, reflect identical sequence
 alterations in genomic DNA. Deletions spanning larger segments of the ATM
 transcript were found to reflect exon skipping, not corresponding genomic
 deletions. This phenomenon usually results from sequence alterations at
 splice junctions or within introns, or mutations within the skipped exons,
 mainly of the nonsense type (Cooper and Krawczak, 1993; Sommer, 1995;
 Steingrimsdottir et al., 1992; Gibson et al., 1993; Dietz and Kendzior,
 1994). One large deletion spans about 7.5 Kb of the transcript and
 represents a genomic deletion of about 85 Kb within the ATM gene. Of these
 deletions and insertions, 25 are expected to result in frameshifts.
 Together with the 5 nonsense mutations, truncation mutations account for
 83% of the total number of mutations in this sample. Nine in-frame
 deletions span long segments (30-124 aa) of the protein, and similarly to
 the truncation mutations, are expected to have a severe effect on the
 protein's structure. It should be noted that two base substitutions
 abolish the translation initiation and termination codons. The latter is
 expected to result in an extension of the ATM protein by an additional 29
 amino acids. This mutation may affect the conformation of the nearby PI
 3-kinase-like domain.
 While the effect of the 5 small (1-3 aa) in-frame deletions and insertions
 on the ATM protein remains to be studied, it should be noted that one such
 deletion (8578del3) leads to a loss of a serine residue at position 2860.
 This amino acid is part of a conserved motif within the PI 3-kinase-like
 domain typical of the protein family to which ATM is related, and is
 present in 7 of 9 members of this family. The single missense mutation
 identified in this study, which leads to a Glu2904Gly substitution,
 results in a nonconservative alteration of another extremely conserved
 residue within this domain, which is shared by all of these proteins. The
 patient homozygous for this mutation, AT41RM, shows the typical clinical
 A-T phenotype. Measurement of radioresistant DNA synthesis in the
 patient's cell line revealed a typical A-T response, demonstrating that
 this patient has the classical A-T cellular phenotype.
 As discussed herein above, the ATM gene of the present invention is
 probably involved in a novel signal transduction system that links DNA
 damage surveillance to cell cycle control. A-T mutations affect a variety
 of tissues and lead to cancer predisposition. This striking phenotype
 together with the existence of "partial A-T phenotypes" endow the study of
 ATM mutations with special significance.
 The ATM gene leaves a:great deal of room for mutations: it encodes a large
 transcript. The variety of mutations identified in this study indeed
 indicates a rich mutation repertoire. Despite this wealth of mutations,
 their structural characteristics point to a definite bias towards those
 that inactivate or eliminate the ATM protein The nature or distribution of
 the genomic deletions among these mutations do not suggest a special
 preponderance of the ATM gene for such mutations, such as that of the
 dystrophin (Anderson and Kunkel, 1992) or steroid sulfatase (Ballabio et
 al., 1989) genes which are particularly prone to such deletions. Thus, one
 would have expected also a strong representation of missense mutations,
 which usually constitute a significant portion of the molecular lesions in
 many disease genes (Cooper and Krawczak, 1993; Sommer, 1995). However,
 only two such mutations were identified in the present study. Other point
 mutations reflected in this series are those that probably underlie the
 exon skipping deletions observed in many patients, again, exerting a
 severe structural effect on the ATM protein.
 A technical explanation for this bias towards deletions and insertions
 could be a greater ability of the REF method to detect such lesions versus
 its ability to detect base substitution. Liu and Sommer (1995) have shown,
 however, that the detection rate of this method in a sample of 42 point
 mutations in the factor IX gene ranged between 88t and 100%, depending on
 the electrophoresis conditions. The 10 base substitutions detected
 directly by the REF method in the present study (Tables 2 and 3), indicate
 that such sequence alterations are detected in our hands as well.
 Since the expected result of most of these mutations is complete
 inactivation of the protein, this skewed mutation profile might represent
 a functional bias related to the studied phenotype, rather than a
 structural feature of the ATM gene that lends itself to a particular
 mutation mechanism. The classical A-T phenotype appears to be caused by
 homozygosity or compound heterozygosity for null alleles, and hence is
 probably the most severe expression of defects in the ATM gene. The
 plethora of missense mutations expected in the large coding region of this
 gene is probably rarely represented in patients with classical A-T, unless
 such a mutation results in complete functional inactivation of the
 protein. By inference, one missense identified in this study, Glu2940Gly,
 which substitutes a conserved amino acid at the PI 3-kinase domain and
 clearly gives rise to a classical A-T phenotype, points to the importance
 of this domain for the biological activity of the ATM protein. Mutations
 in this domain abolish the telomere-preserving function of the TEL1
 protein in S. cerevisiae (Greenwell et al., 1995), a protein which shows a
 particularly high sequence similarity to ATM (Savitsky et al., 1995b;
 Zakian, 1995). Another member of the family of PI 3-kinase-related
 proteins that includes ATM is the mammalian FRAP. Mutations in the PI
 3-kinase domain abolish its autophosphorylation ability and biological
 activity (Brown et al., 1995). These observations, together with the
 mutation shown here, suggest that this domain in ATM is also likely to
 include the catalytic site, which may function as a protein kinase.
 Genotype-phenotype relationships associated with the ATM gene appear
 therefore to extend beyond classical A-T. There are several examples of
 genes in which different mutations lead to related but clinically
 different phenotypes. For example, different combinations of defective
 alleles of the ERCC2 gene may result in xeroderma pigmentosum (group D),
 Cockayne's syndrome or trichothiodystrophy--three diseases with different
 clinical features involving UV sensitivity (Broughton et al., 1994, 1995).
 Different mutations in the CFTR gene may lead to full-fledged cystic
 fibrosis, or only to congenital bilateral absence of the vas deferens
 which is one feature of this disease (Chillon et al., 1995; Jarvi et al.,
 1995). A particularly interesting example is the X-linked WASP gene
 responsible for Wiskott Aldrich syndrome (WAS), characterized by
 immunodeficiency, eczema and thrombocytopenia. Most of the mutations
 responsible for this phenotype cause protein truncations; however, certain
 missense mutations may result in X-linked thrombocytopenia, which
 represents a partial WAS phenotype, while compound heterozygosity for a
 severe and mild mutation results in females in an intermediate phenotype
 (Kolluri et al., 1995; Derry et al., 1995).
 In a similar manner, genotypic combinations of mutations with different
 severities create a continuous spectrum-of phenotypic variation in many
 metabolic diseases.
 Which phenotypes are most likely to be associated with milder ATM
 mutations! Since cerebellar damage is the early and severe manifestation
 of A-T, it is reasonable to assume that the cerebellum might also be
 affected to some extent in phenotypes associated with milder ATM
 mutations. Such phenotypes may include cerebellar ataxia, either isolated
 (Harding, 1993) or coupled with various degrees of immunodeficiency. The
 latter combination has indeed been described, sometimes with chromosomal
 instability, and is often designated "ataxia without telangiectasia" (Ying
 and Decoteau, 1983; Byrne et al., 1984; Aicardi et al., 1988; Maserati,
 1988; Friedman and Weitberg, 1993). Friedman and Weitberg (1993) recently
 suggested a new clinical category of "ataxia with immune deficiency" that
 would include A-T as well as other cases of cerebellar degeneration with
 immune deficits. Evaluation of patients with cerebellar disorders with the
 present invention may reveal a higher frequency of such cases than
 previously estimated. However, in view of the pleiotropic nature of the
 ATM gene, the range of phenotypes associated with various ATM genotypes
 may be even broader, and include mild progressive conditions not always
 defined as clear clinical entities. Screening for mutations in this gene
 in such cases may reveal wider boundaries for the molecular pathology
 associated with the ATM gene.
 Example 4
 Determination of Genomic Organization
 The exon/intron boundaries and the intron sizes were determined using
 long-distance PCR (Barnes, 1994; Cheng et al., 1994; Foord and Rose,
 1994). Primers were designed based on the ATM cDNA sequence (Savitsky et
 al., 1995a,b) at 200-300 bp intervals. Templates for these reactions were
 cosmid and YAC clones, and human genomic DNA. PCR products were obtained
 in all cases, including those that span the largest intron, of 11 kb. In
 the large majority of cases, PCR products of the same size were obtained
 with all templates, and those obtained from genomic DNA were used for
 sequencing of the exon-intron junctions. Following initial reactions, new
 primers were designed as needed, based on the evolving knowledge of the
 gene structure. Exon-intron boundaries were determined at the sites where
 genomic and cDNA sequences diverged. Typical splice acceptor and donor
 sequences were found around these sites in all cases. During the search
 for the A-T gene, six exons were isolated by exon trapping (Shiloh et al.,
 1994b) using the vectors pSPL3 (Church et al., 1994) and eGET (Nehls et
 al., 1994a,b). Their boundaries coincided with those obtained by
 long-distance PCR.
 The ATM gene is composed of 66 exons (FIG. 3 and Tables 1 and 5). The first
 two exons are alternatively spliced, and are designated 1 a (SEQ ID No:10)
 and 1b (SEQ ID No:9). With the exception of the 3' exon, ATM exons (SEQ ID
 Nos:11-63) range in size from 64 to 372 bp, with an average of 149 bp. The
 introns vary considerably in size, from 100 bp to about 11 kb, with the
 majority in the range of 1-3 kb. The consensus dinucleotides GT and AG
 were found at the donor and acceptor splice sites of all introns, except
 for a variant donor site with a GC dinucleotide (reviewed in Jackson,
 1991) present in the intron 3' to exon 51. The first methionine of the
 open reading frame is located in exon 3, whereas the stop codon is located
 in the 3' and largest exon of 3.6 kb. This exon includes a 3' untranslated
 region (UTR)(SEQ ID No:8) of about 3800 nucleotides.
 The ATM gene contains one of the largest number of exons reported to date
 for a human gene. However, these exons are spread over a relatively
 compact genomic region of about 150 kb. The dystrophin gene, for example,
 consists of 79 exons spanning 2.4 Mb of genomic DNA (Roberts et al.,
 1993), while the Huntington's disease gene consists of 67 exons spread
 over 180 Kb (Ambrose et al., 1994b).
 Throughout this application various publications and patents are referenced
 by citation or number. Full citations for the publications referenced are
 listed below. The disclosures of these publications in their entireties
 are hereby incorporated by reference into this application in order to
 more fully describe the state of the art to which this invention pertains.
 The invention has been described in an illustrative manner, and it is to be
 understood that the terminology which has been used is intended to be in
 the nature of words of description rather than of limitation.
 Obviously, many modifications and variations of the present invention are
 possible in light of the above teachings. It is, therefore, to be
 understood that within the scope of the appended claims, the invention may
 be practiced otherwise than as specifically described.