Novel mutations and defective protein kinase C activation of T-lymphocytes in ataxia telangiectasia

Abstract

Three ataxia telangiectasia (AT) patients have been characterized immunologically and molecularly. Patient 1 presents two nondescribed splicing mutations which affect exons 15 and 21 of the ATM gene. The maternal defect consists of a G > A transition in the first nucleotide of the intron 21 donor splicing site which results in a complete deletion of exon 21. The paternal mutation consists of an A > C transversion in the intron 14 acceptor splicing site which produces a partial skipping of exon 15. Two abnormal alternative transcripts were found, respectively, 17 and 41 nucleotides shorter. Patient 2 presents a homozygous genomic deletion of 28 nucleotides in the last exon of the gene. This deletion changes the normal reading frame after residue 3003 of the protein and introduces a premature stop codon at residue 3008 that could originate a truncated ATM protein. Patient 3, a compound heterozygote, presents a defect which consists of a G > A transition in the first nucleotide of intron 62 donor splicing site which results in a complete deletion of exon 62. The results obtained during a three year period in the proliferation assays show an impaired PMA (phorbol myristate acetate) activation in specific T lymphocyte activation pathways (CD69, CD26, CD28, CD3, PHA, PWM and Con A mediated) but not in others (CD2, ionomycin, and Ig surface receptor). The possible link among specific ATM mutations and abnormal immune responses is unknown.

INTRODUCTION

Ataxia telangiectasia (AT) is a high pleiotropic, autosomal recessive disorder characterized by progressive cerebellar ataxia, oculocutaneous telangiectasias, immunodeficiency, premature ageing, high α-fetoprotein serum concentrations and chromosomal instability [1–4].

The incidence of AT has been estimated at 1 in 40000–100000 live births and mutated gene frequency is believed to be very common (1–3%) in the population [5]. The mutated gene was mapped by linkage analysis at 11q23.1 [6] and identified by positional cloning [7]. It spans 184 kb of genomic DNA, producing an mRNA of 13 kb with an open reading frame of 9168 nucleotides [7,8]. The large predicted protein of 3056 residues has a carboxy region of 350 amino acids that has a PI 3-kinase ‘like’ domain, which could be involved in DNA damage processing and cell cycle control [9,10]. ATM interactions with c-Abl, p53 and the recently described Karp-1 support its function in cellular response to DNA damage [11–14].

The pleiotropic nature of the clinical and cellular phenotype suggests that the ATM protein plays an important role in maintaining the stability of the genome but also has a more general role in signal transduction [15]. The ATM protein appears predominantly in the nucleus of human fibroblasts, but is present in both the nucleus and cytoplasm in human lymphoblastoid cell lines and peripheral blood lymphocytes [15,16]. In fact, AT lymphocytes show a defect in the cytoplasm-to-nuclear signalling, demonstrated by the defective lymphocyte proliferation in response to several membrane stimuli continuously recorded in AT patients [4]. It has been demonstrated that ATM and p53 could participate in common PKC mediated signalling pathways, since the ionizing radiation-induced increase in the p53 protein is both delayed and reduced in extent in AT cell lines and specific PKC inhibitors block the increase in p53 [17–19]. Recently, it has also been found that ATM and PKCdelta are common novel targets of caspases. The functional significance of the ATM cleavage is supported by the finding that expression of ATM protected in part against apoptosis [20].

The ATM gene cloning has favoured AT mutation screening. A diverse array of mutations, most of them leading to truncated proteins, has been described (http://www.vmresearch.org/atm.htm). Most of the patients are compound heterozygotes and consanguinity has been demonstrated in most homozygous patients. A significant number of the mutations described affects the mRNA splicing driving to exon skipping [7,21–25].

In this report, PBMCs from three AT cases and a T-cell line immortalized with HVS from one of the patients have been characterized on the molecular, immunological and cytogenetic levels. The immune status follow-up of the three patients shows impairment in the lymphocyte proliferative responses induced by PMA, that is an analogue of diacylglicerol and a specific direct activator of the PKC pathway [26].

PATIENTS AND METHODS

Patients

The patients were diagnosed of AT by clinical criteria (all of them were suffering from cerebellar ataxia, oculocutaneous telangiectasias and persistently elevated α-fetoprotein peripheral levels). Patient 1 is a 9-year-old Spanish boy from non-cosanguineous parents. At the age of 7 years, pronounced alterations in his cellular immunophenotype were detected and responses to ‘in vitro’ challenging with different mitogens were reduced, although he did not present severe infections or malignant tumours. The immunological status of the patient has been studied three times in the last 24 months. Patient 2, who has 5 healthy siblings, is an 8-year-old Spanish girl who suffered several episodes of pneumonia with hospitalization. She is also affected by the Goldenhar syndrome (oculoauriculovertebral dysplasia) [27], and was admitted to our hospital with pronounced alterations in immunoglobulin levels, an altered cellular immunophenotype, and severe malnutrition. Patient 3, a 9-year-old Spanish boy, has the clinical AT features with repeated respiratory infections and otitis, IgA deficit and moderate hyper-IgM.

Cell lines

T lymphoblastoid cell lines were established by transformation of PBMCs from the patients and healthy age-matched controls with HVS supernatant. This was obtained from cultures of an owl monkey kidney cell line (OMK cell line, American Type Culture Collection, CRL 1556) lytically infected with HVS strain C488 and transformations were performed as previously described [28].

Quantification of serum proteins

Immunoglobulin levels (IgG, IgA, and IgM) and complement factors (C3 and C4) were measured by nephelometry (Array 360 system, Beckman, Brea, CA). Serum haemolytic capacity (CH100) and serum IgE and IgG subclasses were measured by radial immunodiffusion (The Binding Site, Birmingham, UK). α-fetoprotein was assayed by nephelometry.

Proliferation assays in PBMCs

8 × 10⁴ cells were placed in round-bottomed microtitre plates (Nunc, Roskilde, Denmark) in 170 µl of AIM-V culture medium (Gibco BRL, Paisley, UK) supplemented with 1% penicillin/streptomycin (Difco) and 1% glutamine 20 mm (Whittaker, Walkersville, MD). The following stimuli or their combinations were used in triplicate [29]: anti-CD3 (OKT3) at 12·5 ng/ml (Ortho Pharmaceuticals, Raritan, NJ); anti-CD28 at 50 ng/ml and anti-CD2 (1·1 + 2·1) 1 : 40 final (CLB Plesmanlaan, Amsterdam, the Netherlands); anti-CD26 at 200 ng/ml (Coulter Clone, Hialeah, FL); anti-CD69 at 7·5 µg/ml (Becton Dickinson, Sunnyvale, CA); PHA (1 : 100 final) and PWM 1% v/v (Difco); enterotoxin A and enterotoxin C1 at 1 ng/ml (Serva, Heidelberg, Germany); PMA 10 ng/ml (Sigma, St. Louis, MO); ionomycin 1 µm and Protein A from Staphylococcus aureus (Pansorbin), 1 : 500 final, and ConA 0·3 mg/ml (Calbiochem, La Jolla, CA); rIL2 50 U/ml (Genzyme, Boston, MA). Wells were individually pulsed with 1 µCi of [³H]thymidine after 3 days of culture and its uptake was measured in a liquid scintillation counter (1205-Betaplate; Pharmacia LKB-Wallac, Turku, Finland). Data were normalized as the net percentage of cpm obtained with a given stimulus relative to the maximum stimulus in the same assay.

Cytogenetic studies

Chromosome preparations and Giemsa-stained metaphases were obtained by standard methods [30] from PHA-stimulated HVS cells or from PBMCs. FISH on chromosomes 7 and 14 for patient 1 was done by standard methods [31] using digoxigenin-labelled total chromosome probes (Oncor, Gaithersburg, MD).

Cytofluorographic analysis

For direct immunofluorescence, 1 × 10⁶ cells were incubated for 30 min at 4°C using the corresponding monoclonal antibodies as described previously and an EPICS-XL flow cytometer [32].

RNA and DNA amplification

Cytoplasmic RNA extracted from cultured cells or PBMCs using the NP-40 lysis method [33] was reverse transcribed and amplified with specific primers (using a one-step RT-PCR method; Gibco BRL), following the manufacturer's instructions. These primers were partially overlapping and the sizes of the PCR products ranged from 1019 to 1541 nucleotides, covering all of the ATM coding region. The primers used were: AT(− 144–1104) (a) 5′-TACTACTTTGACCTTCCGAGTGC-3′ and (b) 5′-TTGAGAAA TCTCCAAGGATCTGG-3′; AT(1001–2198) (a) 5′-CAGGATTT CGTAATATTGCCG-3′ and (b) 5′-CCCATGTAACAGTAGCA GCC-3′; AT(2029–3570) (a) 5′-AGTATTGGCTTCTCTGTCCA CC-3′ and (b) 5′-CACAAGGTGAGGTTCTAATCC-3′; AT(3461 –4992) (a) 5′-TACTGACGTTGATAGCTGTGG-3′ and (b) 5′-TTCACCAGTGTGGTTTATTGC-3′; AT(4914–6198) (a) 5′-TC CGCAAGATGGGATTATGG-3′ and (b) 5′-CTGAATGATTCC TGCCTGGCG-3′; AT(6043–7062) (a) 5′-CCAGATAGTTTGTATGGCTGTGG-3′ and (b) 5′-CGCAGGATTTTCTAAGCAC G-3′; AT(6952–8081) (a) 5′-AAGTTGGATGCCAGCTGTGC-3′ and (b) 5′-CCTGCTAAGCGAAATTCTGC-3′; AT(7910–9124) (a) 5′-AGTGGAAGACTCAGAGAAAAGGC-3′ and (b) 5′-GGT CTATGGCCTGCTGTATGAGC-3′. Additional primers were used for mutation analysis: AT(2635–3022) (a) 5′-TAGGTGCCA TTAATCCTTTAGC-3′ and (b) 5′-GAGTCCATATTGCTTTGA CCTAGG-3′; AT(1807–2081) (a) 5′-TTTCC TCATCTTGTAC TGGAG-3′ and (b) 5′-AGACAGCGATCCAG TGATTCC-3′; AT (8952–9124) (a) 5′-GAATGCAGATGACCAAGAATGC-3′ and (b) 5′-GGTCTATGGCCTGCTGTATGAGC-3′; and AT(8619–8898) (a) 5′-CAGAATATCTTGATAAATGAGC-3′ and (b) 5′-CTTTCAAAGGATTCATGGTCC-3′. DNA or RNA amplification reactions were carried out as described previously [33].

REF analysis and DNA sequencing

1 µg of the PCR product was digested separately with five restriction endonucleases. Denatured and nondenatured PCR digestion products were subjected to electrophoresis in 6–10% polyacrylamide gels [34] using the Protean II vertical electrophoresis system (20 × 20 cm gel size; Bio Rad Laboratories, Hercules, CA), and the gels were silver stained (Bio Rad). The products for sequencing were purified by using the QIAquick gel extraction kit (QIAGEN, Hilden, Germany) and were cloned in the pMOS-Blue vector (Amersham, Buckinghamshire, UK). Double-stranded DNA sequencing was done as previously described [35]. Each sequence was confirmed by analysis of three or more clones from two different amplifications.

RESULTS

Humoral immunity and T-cell phenotype are dramatically altered in AT (Table 1)

Table 1.

Humoral immunity and lymphocyte phenotype in the AT patients^*

	Patient 1	Patient 2	Patient 3	Reference values
Humoral immunity (expressed as mg/dl)
IgA	159/167/126	65/–/119	< 7/ < 7/ < 7	57–204
IgM	140/169/120	258/–/334	577/627/456	44–242
IgG2	–/86/77	< 68/–/158	72/363/308	113–480
PBMCs phenotype (expressed as percentage of positive cells)
Lymphocytes
(absolute number/µl)	1711/940/844	1489/893/1845	2200/1650/1907	1652–4049
T cells
CD28	36/–/31	26/33/26	37/36/44	40–65
CD3	54/55/48	40/54/42	48/57/60	59–77
CD4	22/26/23	15/18/15	29/25/32	29–49
CD4 naive	2/–/2	4/2/2	3/1/4	16–39
CD4 memory	19/–/20	13/14/12	26/22/31	5–15
CD8	22/18/17	25/34/25	16/21/16	11–37
TcRαβ	–/47/25	–/45/–	–/–/–	55–70
TcRγδ	16/12/11	1/4/2	–/5/2	2–7
B cells
CD19	9/8/7	6/5/3	38/31/28	8–23
NK cells
CD16	28/34/39	45/34/48	8/12/8	3–18

^*The following parameters were unaltered in the patients compared to controls: IgG, IgG1, IgG3, IgG4, C3, C4 and CH100 peripheral blood concentrations or activity. CD2, CD18, CD43, CD5, CD7, CD57, and CD14 PBMCs subpopulations. Reference values were obtained from 100 unrelated healthy children under 14 years. –, Not determined. Bars separate results obtained in different studies.

At least two studies with a minimal interval of six months were done for each patient. The immunoglobulin levels in patient 1 were within the normal values in three studies (with only a mild IgG2 deficiency). Patient 2 showed mild hyper-IgM and low values in some studies for IgA and IgG2. Among patients, the pattern of humoral immunity was most altered in patient 3 with hyper-IgM and an IgA deficit. These heterogeneous results agree with previously published data on the humoral immunity of AT patients [1–4].

Lymphocyte phenotypes, especially the T-cell phenotype, are severely altered. All three patients showed decreased amounts of total lymphocytes which were often below the reference values. Except in one study of patient 3, all the patients presented low percentages of CD3⁺ T-cells due to low CD4⁺ T-cells levels, since all of them showed normal CD8⁺ T-cell numbers. Total CD4⁺ T-lymphocyte reductions were caused by a dramatic reduction of the naive subset, whereas memory CD4⁺ cells were within the normal range or even slightly increased [36]. In patient 1 total CD3⁺ cells decreased from 54% to 48% over time, probably due to the slight decrease observed in CD8⁺ and TcRγδ⁺ subsets. A dramatic reduction of TcRαβ⁺ cells was also observed and inversely correlated with a substantial increase in the natural killer cells (CD16⁺ from 28% to 39%). It is interesting to emphasize that both patients, 1 and 2 have very low levels of B cells (CD19⁺) and on the contrary both possess increased levels of natural killer cells (NK, CD16⁺) in contrast to that observed in patient 3.

Proliferative responses of lymphocytes are severely impaired in AT (Table 2)

Table 2.

Lymphocyte function in the AT patients^*

	Patient 1	Patient 2	Patient 3	Reference values
Normal responses (+ PMA)
Protein A of S. aureus	3/1/4	–/3	3/8/12	9–31
Protein A + PMA	7/2/8	–/8	38/17/35	5–28
Ionomycin + PMA	55/21/27	–/42	72/70/65	27–95
α-CD2 + PMA	81/100/95	10/28	45/38/61	33–94
Moderately impaired responses (+ PMA)
α-CD3	27/5/52	23/62	37/65/36	21–32
α-CD3 + PMA	15/25/100	12/25	41/21/38	56–86
PHA	32/1/10	48/86	3/30/21	29–78
PHA + PMA	34/20/68	10/36	18/27/37	59–76
α-CD28 + PMA	12/9/61	6/9	20/5/18	13–100
Severely impaired responses (+ PMA)
Con A	3/1/8	13/12	1/7/9	23–64
Con A + PMA	1/0/5	2/7	3/5/9	29–94
PWM	10/12/18	7/30	24/32/19	27–37
PWM + PMA	9/14/19	–/9	16/8/10	32–54
α-CD26 + PMA	19/2/14	–/17	6/7/11	30–98
α-CD69 + PMA	1/0/6	–/4	2/3/6	11–50

^*Patients and reference results are expressed as net relative percentage of each stimulus to the maximum proliferation obtained among all the stimuli used for each individual (see Discussion). Cellular proliferation with CD2, ionomycin, CD28, CD69 and CD26, used independently, is comparable to negative control. Reference values were obtained from healthy controls assayed in parallel and are expressed as the range of response obtained in 8 different studies. –Not determined. Bars separate results obtained in different studies.

The patients' PBMCs presented decreased proliferative responses to most of the stimuli assayed in the ‘in vitro’ functional evaluation regarding DNA synthesis (net cpm). The patients' proliferative responses to IL2, CD2, CD3, CD28, PHA, enterotoxin A, PMA, PHA + IL2, CD2 + CD28, CD3 + IL2, CD2 + IL2, and Con A + IL2 were within normal values. Con A induced proliferation was repeatedly reduced in all studies. When PMA was used as a costimulus with antigens, monoclonal antibodies or lectins, a differential pattern was observed: (a) anti-CD2, ionomycin, and protein A (all three in combination with PMA) triggered normal responses; (b) PMA in combination with anti-CD28, anti-CD3 or PHA showed moderate impairment (with no additional induction or mild inhibition of the responses induced by PHA, anti-CD3 or anti-CD28 alone); and (c) PMA combinations with anti-CD69, anti-CD26, Con A or PWM were severely impaired.

HVS T cell analysis of patient 1

6 months after HVS infection, the T cell line was stable. HVS DNA was detected in T cells by PCR (data not shown). Immunophenotype analysis showed that 100% of T cells were CD3⁺, CD8⁺, CD45R0⁺ (memory cells) and 100% were activated (CD69⁺, CD26⁺, CD56⁺). However, the T cells did not express CD80 or CD28 antigens. Interestingly, TcRαβ staining showed a dull expression at the beginning of the culture (3 months) that turned to a bright expression when the cells were stable (6 months). This phenomenon was not observed in the healthy control cell line (data not shown).

High incidence of spontaneous chromosome breaks

Karyotype analysis in PBMCs of patients 1 and 2 showed a great increase in spontaneous chromosome breaks (20–30%, respectively) when compared to a healthy control (<1%) as previously described for AT patients [37]. The breaks were mostly nonspecific and included a chromosome 7 paracentric inversion (inv(7)(q14; q35)). Moreover, G banding technique was assayed on well-established HVS T-cell lines of patients 1 and 2, and a balanced translocation involving chromosomes 7 and 14 was observed in 100% of the cells in patient 1 (Fig. 1). FISH techniques made it possible to determine precisely the translocation points to t(7; 14)(p14; q12) (Fig. 1), one of the most frequent translocation events observed in other AT patients [37]. On the other hand, patient 2 showed, as far as we know, a previously undescribed balanced translocation which involves chromosomes 4 and 14 and affects the TcRα loci (t(4; 14)(p14; q12)) in 100% of the T-lymphoblastoid cells.

Fig. 1.

Ideograms, G-banding patterns and FISH of both (A) normal chromosomes 7 and 14 and (B) translocated (7; 14) chromosomes observed in the HVS-T-cell line of patient 1.

Mutation analysis and identification (Table 3)

Table 3.

Summary of the ATM gene mutations detected in three Spanish AT patients

Patient	Genotype*/ Origin	Genomic mutation	Effect on cDNA	Intron/ Exon	Codon change	Consequence
1	Compd Htz/Maternal	IVS21 + 1G > CA	2839del 83	21	Frameshift	Exon 21 skipped, stop at residue 955 [21]
1	Compd Htz/Paternal	IVS15–2A > C	1899del 17	15	Frameshift	Stop at residue 632
			1899del 41
2	Homozygous/	9008del28	9008del28	65	Frameshift	Stop at residue 3008
	–
3	Compd Htz/	IVS62 + 1G > A	8672del 115	62	Frameshift	Exon 62 skipped, stop at residue 2899 [24–25]
	–

^*Compd Htz, compound heterozygote

The ATM cDNA was scanned for mutations in both PBMCs and the T-cell lines. The analysis was based on RT-PCR followed by a modification of REF [34]. The coding sequence of the ATM mRNA was divided into eight partially overlapping fragments, and each fragment was analysed separately. Mutations identified by this assay were confirmed by repeating the RT-PCR and in genomic DNA.

Maternal mutation for patient 1 consisted in a large deletion observed in the third fragment [AT(2028–3569)] of the ATM cDNA. A new RT-PCR reaction using an internal primer set AT(2635–3022), showed a total lack of exon 21. Genomic DNA amplification of exon 21 in the patient, the patient's family and a control, using a pair of primers previously published [38], showed a specific change (G > A) in position + 1 (relative location to exon 21) of intron 21 (Fig. 2). This mutation in the intron 21 donor site resulted in an aberrant splicing that joins exon 20 to exon 22. As a consequence of the 83-bp deletion, the normal reading frame is changed after Met 946, producing a premature stop codon at residue 955.

Fig. 2.

Schematic representation of maternal (GenBank accession numbers: AF035325 for genomic DNA and AF035328 for cDNA) and paternal-inherited mutations (GenBank accession numbers: AF035324 for genomic DNA and AF035326 and AF035327 for cDNAs) of patient 1. The location of relevant introns and exons of genomic DNA in the ATM gene is shown in the upper panel followed by both the normal and the mutated DNA sequences. Lowercase letters indicate introns and uppercase letters indicate exons. Donor and acceptor splicing signals are boxed. A vertical arrow depicts the genomic mutation. Double arrow horizontal lines show the normal splicing sites and the alternative abnormal splicing in the patient for the maternal-inherited mutation. Two putative novel acceptor sites inside exon 15 are boxed for the paternal-inherited mutation.

The paternal mutation for patient 1 was localized in the restriction component of the REF assay [34]. Differences between the control and the patient were observed in the second fragment of ATM cDNA [AT(1000–2197)] (results not shown). Internal primers, AT(1807–2081), were designed to amplify a smaller region in the mRNA which included exon 15; furthermore a pair of intronic primers [38] were also used in genomic DNA. In fact, cDNA amplification showed the presence of three different fragments in the patient, one of them the same size as that of the cDNA control and two smaller. All three cDNA fragments as well as the genomic exon 15 DNA in the patient, patient's parents and a healthy control were cloned. A point mutation in nucleotide − 2 (relative location to exon 15) of the ATM gene A > C was found in the patient and his father. This mutation causes the loss of the normal acceptor site for the intron 14 splicing. Clones corresponding to two alternative splicing sites located 3′ downstream the mutation and inside exon 15 (resulting in the loss of 17 and 41 nucleotides of exon 15, respectively) were detected (Fig. 2). Both alternative splicings introduce a premature TGA stop codon and therefore might lead to a truncated protein (Fig. 2).

For patient 2, an RT-PCR experiment showed a deletion in fragment eight of the ATM gene [AT(7910–9124)], which seemed to be in homozygosity. The breakpoints of the deletion were delimited by a restriction endonuclease analysis (results not shown) and further direct sequencing with a new RT-PCR reaction using an internal primer set AT(8952–9124). DNA and mRNA sequencing showed a deletion of 28-bp (Fig. 3).

Fig. 3.

Schematic representation of patient 2 homozygous mutation. See legend of Fig. 2 for the meaning of the symbols. A vertical arrow shows boundaries between intron-exon or where the putative protein is changed in the patient. Above the amino acid 3002, the patient would have a normal protein however, this changes from residue 3003 on. The location of relevant portions of ATM cDNA and their corresponding amino acid sequence are shown in the bottom panel. The 5 amino acids changed in the patient's truncated-protein are shown in italics.

Finally patient 3 is a compound heterozygote and at present we have identified a large deletion in fragment eight [AT(7910–9124)] of cDNA. Direct sequencing with a new RT-PCR reaction using an internal primer set AT(8619–8898), which rendered a 279-bp product, showed a total lack of exon 62. Genomic DNA amplification of exon 62 in the patient and a control [38], showed a specific change (G > A) in position + 1 of intron 62. This mutation in intron 62 donor site resulted in an aberrant splicing that joins exon 61 to exon 63. This mutation has already been reported [24,25]. As a consequence of the 115-bp deletion, the normal reading frame is changed after Leu 2890, producing a premature stop codon at 2899 residue that might originate a truncated and probably nonfunctional ATM protein.

DISCUSSION

We report the molecular and immunological function of three Spanish ataxia telangiectasia patients. All of them presented the severe AT phenotype characterized by progressive cerebellar ataxia, oculocutaneous telangiectasias, high serum concentrations of α-fetoprotein, and substantial abnormalities in humoral immunity, immune phenotype and lymphocyte function. Patient 1 has not suffered repeated and serious infections during his life, patient 2 has needed hospitalization on several occasions due to repeated infections and patient 3 has suffered from otitis and repeated pulmonary infections.

The patients studied showed a dramatically altered PBMCs phenotype (Table 1) with a very low level of CD3⁺ cells and an absolute disruption of the CD4⁺ naive/CD4⁺ memory cells ratio and almost exclusively had ‘memory’ cells (contrary to that expected in a healthy child); this disruption in lymphocyte differentiation has been previously described [36]. Both patients 1 and 2 showed a progressive T-cell differentiation difficulty, with a severe TcRαβ⁺ cell reduction and an important NK cell increase. These T cell differentiation problems could be explained by rearrangement failures in the TcR genes, with a probable accumulation of unstable hybrid TcR molecules that abort TcR surface expression. This hypothesis is supported by the high incidence of chromosomal breaks observed in the patient's PBMCs, especially involving chromosome 7, where the TcR β and γ loci are located [37].

The lymphocytes of the AT patients presented decreased responses to most of the stimuli assayed in the ‘in vitro’ functional evaluation in regards to DNA synthesis (net cpm) similar to the previously published data [4]. Data normalization as the net percentage of cpm obtained with a given stimulus relative to the maximum stimulus in the same assay, and the use of serum-free medium (described in ‘Methods’), made it possible to compare results between studies carried out at different times (Table 2). The patients' responses to IL2; MoAbs anti-CD2, anti-CD3, and anti-CD28, to lectins as PHA or to antigens as enterotoxin A were comparable to those obtained in healthy controls. However, the combination of phorbol esters (PMA) with a second stimulus as stimulation cocktail resulted in a severe inhibition of the poor proliferation obtained by the stimuli themselves. Thus, proliferation was severely impaired when PMA was used together with anti-CD69, anti-CD26, Con A or PWM. Proliferation was not affected or was mildly affected when PMA was used together with anti-CD3, PHA or anti-CD28, and PMA improved proliferation when it was used with anti-CD2, ionomycin or protein A. When we used only PMA as a stimulus in the proliferation assay, the response was comparable with the control, showing that PMA is not cytotoxic to the concentration used in our proliferation assay. Further experiments should be carried out to find out why PMA sometimes improves AT lymphocyte activation (through protein A, CD2 and ionomycin activation pathways) and sometimes inhibits proliferation (through Con A, CD69, PWM, CD26, CD28, CD3 and PHA activation pathways). Data obtained in the present study confirms these two opposing actions of PMA on ATM deficient lymphocytes, depending on the triggered biochemical pathway.

Studies carried out in a NBS patient [39] have shown lymphocyte proliferation, especially the one obtained with PMA, and subpopulation patterns similar to those obtained for AT in the present article. Both AT and NBS exhibit very similar clinical and cellular phenotypes and chromosomal abnormalities.

Three different genomic point mutations leading to alternative mRNA splicings which affect coding exons and a genomic homozygous deletion have been characterized in our patients and their parents (Table 3). Neither of the two point mutations are natural polymorphisms of the ATM gene because both provoke drastic effects in the mRNA ATM sequence. Maternal mutation in 1 (at the nucleotide + 1; donor splicing site of intron 21) resulted in the exon 21 loss (83 bp) of the ATM transcript; this mutation has been previously described in a Turkish consanguineous family [21], but the genomic underlying defect was unknown until the present study. Exon 21 has 83 nucleotides and its loss produces a change in the normal open reading frame and a protein truncation at codon 955 (Fig. 2). Patient 3 is a compound heterozygote and presents a mutation (G > A) at the nucleotide + 1 of the donor splicing site of intron 62, which provokes the skipping of exon 62 and therefore, the generation of a premature stop codon. This mutation has been described in heterozygosity in two previous works [24–25].

The paternal mutation of the patient 1 has not been described previously and it consists of an A > C substitution at position − 2 in the genomic intron 14 acceptor-splicing site. This change leads to the loss of the intron 14 consensus acceptor site. The AT patient's cell splicing machinery uses two alternative acceptor splicing sites, both of them located inside the exon 15, rendering shortened (17 and 41 nucleotides, respectively) processed mRNAs; both shortened transcripts carry null mutations because of the introduction of a premature in-frame stop codon (Fig. 2). Most of the cDNA clones obtained and the higher intensity in the RT-PCR experiment demonstrate that the shortest alternative processed mRNA is preferentially used ‘in vivo’, probably because the splicing site used (tctcag) is more similar to the native one (ttgaag) and more similar to the eucariotic consensus splicing sites [40]. In fact, the SIGNAL software program (PC GENE, release 6·85, Intelligenetics, Mountain View, CA) assigned this sequence the greatest probability of being the new splicing site. Alternative mRNA splicing has been usually associated with the use of different 5'promoter elements in the ATM gene [7] or with the use of different 3′ polyadenylation signals rather than with the introduction of premature stop codons, as observed in the paternal mutation. However, alternatively spliced exons coding for premature stop codons, which lead to truncated proteins, has been previously described [41]. Truncation often occurred at the 3′ end region of the gene [22].

It is interesting to emphasize that ATM mRNAs carrying serious mutations (introduction of a stop codon, aberrantly spliced mRNAs, etc.) are present in AT cells and are apparently stable; this feature has also been described for other defective genes [41]. However, the normal behaviour of cells is to eliminate these abnormal messages [42], probably in an attempt to save energy and thus avoid the translation of proteins that are not going to have any function. The classical AT phenotype is caused mostly by ATM null mutations [3,7,21–25], and truncated proteins are generally eliminated from the cells [23,43], probably due to their instability.

AT homozygotes and compound heterozygotes show nonrandom chromosomal aberrations in lymphocytes, such as translocations and inversions, which preferentially involve chromosomal breakpoints at 14q11, 14q32, 7q35, 7p14, 2p11 and 22q11 [37]. Interestingly, the breakpoints of these rearrangements correspond to chromosomal bands which bear immunologically relevant genes (T-cell receptor α, β, and γ chain and the immunoglobulin heavy-chain are localized near the breakpoints). This could explain the ‘dull’ expression of TcR complexes on the HVS-T cell line surface at the beginning of the culture in the patient 1 (data not shown). Thus, HVS transformed T-cell lines in AT patients could be used in the near future as a target for ‘in vitro’ gene therapy experiments, in the same way as EBV lymphoblastoid cells had been used in other gene therapy trials [44] and also to follow up AT patients immune function more accurately by testing cells from previous drawings of blood together with currently obtained T-lymphocytes. HVS transformed T-cell lines have also been found useful in other primary immunodeficiency research as the CD3γ deficiency [28] and the Wiskott–Aldrich syndrome [45].