Ataxia-telangiectasia: A multiparameter analysis of eight families

(‘1 INICAIL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

Ataxia-Telangiectasia:

23, 501-516 (1982)

A Multiparameter Eight Families**’

Analysis

of

RICHARD A. GATTI,~ MIRIAM BICK, CHICK F. TAM, MICHAEL A. MEDICI, VIVI-ANNE OXELIUS, MATTHEW HOLLAND, ALLAN L. GOLDSTEIN, AND ELENA BODER Depurtment of Pediatrics, Cedars -Sinai Medical Center, Los Angeles, California 90048: University of Lund, Department of Pediatrics, Lund, Sweden; the Department of Pathology. UCLA School of Medicine, Los Angeles, California 90024; and the Depurtment of Biochemistry. George Washington University. Washington D.C. 20006

Eight families, including 12 patients with ataxia-telangiectasia (AT), were investigated for parameters which tested the current models of pathogenesis. We found: T cells decreased, B cells elevated, PHA responses decreased, variable T suppressor activity, serum IgA and IgGz decreased. AFP and liver enzymes in serum were elevated. In addition, several parameters related to cell membrane and cytoskeletal functions were abnormal: increased concanavalin A capping and increased cyclic nucleotides of lymphocytes and decreased chemotactic responses of neutrophils. Thymosin-a-l levels of patients and family members did not differ from age-matched controls. While many of the immunological abnormalities seen in AT could be due to a common immunoregulatory defect, this would not explain the neurological deterioration or the DNA aberrations. We suggest that a broader mechanism be considered in future analyses, such as the possibility that a primary or secondary cytoskeletal disorder might underlie the myriad abnormalities seen in AT.

INTRODUCTION

Over the past 4 years, we have had the opportunity to investigate the pathogenesis of ataxia-telangiectasia (AT) by performing investigations on a group of 12 patients and family members. These studies initially focused on characterizing the immune status of these patients and families. They were later expanded to probe for possible evidence of an underlying membrane dysfunction or cytoskeletal abnormality. METHODS

Eight families were studied, including 12 patients, 13 parents, and 6 siblings. For most parameters, the data were analyzed in four groups: patients, parents, siblings, and controls. For parameters which might be age dependent, an agematched control group of approximately 12 children from 8 to 14 years old was added (i.e., “
occasion of his 60th birthday. 28631, CA 16042, and AG 00790, Children’s the Alice and Julius Kantor Charitable Trust. of Pathology, Los Angeles, CA 90024.

501 0090-1229/82/050501-16$01.00/O Copyright @ 1982 by Academic Press, Inc. AU rights of reproduction in any form reserved.

502

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AL.

with a cluster of 8 patients between 8 and 12 years old (8,8,9,9, 10, 12, 12, 12,20, 22, 23, 28). Ethnic background of the families included: Australian, North American and European Caucasian, Ashkenazi Jew, and Central American Spanish. The diagnoses were ascertained by one of the authors (E.B.). All patients have been followed for at least 3 years since diagnosis. None were receiving treatment at the time of study. Lymphocytes were isolated from peripheral blood which was anticoagulated with preservative-free heparin (1000 IU/ml). They were separated over Ficoll-Isopaque gradients. Neutrophils were collected by dextran (3%) sedimentation of the pellets. All cellular parameters were measured on fresh specimens. E-rosetting was performed in a standard assay, utilizing neuraminidase-treated sheep erythrocytes (En). Surface membrane immunoglobulin (SmIg) and Con A capping were determined by direct immunofluorescence, using commercial antisera and Ploem illumination as previously described in detail (1). Antisera for T and B lymphocytes were used in an indirect immunofluorescence assay. Capping was measured as follows: to a pellet of 2 x log cells, 50 ~1 of fluoresceinated Con A (diluted 1: 10) was added. Pellets were resuspended and incubated at 4°C for 30 min. The cells were washed thrice with Hanks solution, resuspended in 0.5 ml of RPM1 1640 and incubated for 90 min at 4” or 37°C. After incubation, the cells were washed thrice, resuspended in 90% buffered glycerol, and examined with a Leitz Ortholux microscope. One hundred fluorescein-positive cells were counted and grouped into three categories: (i) noncapping; (ii) incomplete capping or patching; and (iii) complete (active) capping, i.e., when all fluorescein positivity was observed in less than one-fourth of the cell circumference. The cells were counted without knowledge of which specimens belonged to patients, family members, or controls. c-w-Naphythyl acid esterase (ANAE) were measured by first E-rosetting lymph@ cytes, cytocentrifuging the preparation onto glass slides, air-drying, and staining (2). We modified the staining procedure slightly by adjusting the pH to 5.8 to minimize precipitation of the stain. We also found that a 4-hr incubation period (at 20°C) stabilized staining intensity. All counts were performed by the same individual. En+ cells with at least one focal, distinct red-brown reaction “dot” were scored as positive; cells with diffuse or no staining were scored as negative. Cells without adhered sheep erythrocytes were not scored. At least 200 En+ cells were examined on each slide. Levels of En+/ANAE+ cells in PBL of normal donors vary little from one laboratory to another (3-5). Phytohemagglutin (PHA) responses were measured in a standard assay (1). Lymphocytes, 1 x 105, in 0.2 ml RPM1 with 10% AB serum, L-glutamine, and antibiotics added, were stimulated with four concentrations of PHA, so as to include two optimal and two suboptimal points. Cultures were performed in triplicate. DNA synthesis was measured by incorporation of tritiated thymidine and expressed in counts per minute. The maximum response from each curve, regardless of which concentration, was compared to the maximum response of the daily control(s) and the results reported as a percentage of the control(s). T suppressor activity was measured by first inducing T suppressor cells in the patients’ lymphocytes with Con A (l:lOOO)-or without Con A as a sham

ATAXIA-TELANGIECTASIA

503

control-harvesting after 48 hr, irradiating with 2000 rad, and then coculturing these cells for 48 hr with normal lymphocytes which were stimulated with Con A (1:500 and 1: 1000). The results were calculated as follows:

Ts =

(T sham [Con A] - T sham [no Con A]) (T supp [Con A] - T supp [no Con A]) T sham [Con A] - T sham [no Con A]

x 100.

In this way, we hoped to minimize nonspecific stimulation from either allogeneic antigens or extraneous antigens in the tissue culture medium. Neutrophil chemotaxis was performed using Neuroprobe blind-well chemotactic chambers, with SSWP Millipore filters separating the cells from either McCoy’s with 10% fetal calf serum or from McCoy’s with 10% fetal calf serum and the chemotactant, N-formyl-methyl-leucyl-phenylalanine (MLP) (customsynthesized by Chemical Dynamics Corp., South Plainfield, N.J.). A dose-response curve to three concentrations of MLP (10p7, 5 x lo-*, 1O-8 M) was used. Neutrophils completely transversing the filter were counted at 400 x by averaging the number of cells per 10 fields. Random mobility was assessed by measuring neutrophil movement across the filter with no chemotactant in the lower well of the chemotactic chamber. Chemotactic indices were calculated by dividing maximum number of cells responding to the chemotactant by the random mobility control. All tests were run in triplicate. Cyclic nucleotides were assayed on En-rosette-enriched lymphocyte preparations as described previously (6). Levels were expressed as picomoles per lo7 lymphocytes. Specimens were coded with regard to their groups. Each AT specimen was “matched” with a normal control of comparable age. Thymosin-a-1 was measured by a radioimmunoassay (7) on coded sera which included age-matched and daily controls. IgG subclasses were measured by an electroimmunoassay on similarly coded sera (8). Results were compared to those of previously studied age-matched populations (9). a-Fetoprotein was measured in a commercial laboratory (Bioscience Laboratories, Van Nuys, Calif.) using a radioimmunoassay. RESULTS T and B Lymphocyte

Levels

The data depicted in Fig. la confirm many previous reports of depressed T-cell proportions, as measured by E-rosette formation, in peripheral blood lymphocytes (PBL) of AT patients (P < 0.05), and also show that the mean T-cell level for parents (68%) is intermediate between those of patients (55%) and controls (80%). The mean E-rosette values for siblings was 72%. T-cell proportions were simultaneously measured utilizing a mouse monoclonal anti-T antiserum (3Al) which identifies primarily T helper cells [courtesy of A. Fauci (lo)]. Levels in four patients were low; however, they were not as low with the monoclonal antibody marker as with E-rosetting and differences between the patient and control populations were marginally significant (P = 0.01). This suggests the presence of immature T cells in PBL of AT patients which do not rosette

504

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%

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%

Normal Adult Values

60

20

ET

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Anti-T

80

60

1 I

I

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I

I

I

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AT

Parents

Sibs

AT

Parents

Sibs

Controls

* Fauci’s

3Al

a

“Ant!-B”Antiserum*

Smlg % 50

%

50

a 0

40 00

T

30

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1

I

Parents

Sibs

Normal Adult Range

20

AT

I

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1

Parents

Sibs

Controls

*Strommgeis

Anti-p23.30

b

FIG. la. FIG. lb. cytes and (see text),

T-cell levels. B-cell levels. both kappa+ but correlated

Note low levels in patients and intermediate levels in parents. Note elevated levels in patient group. The patient with 48% SmIg+ lymphoand lambda+ cells. The p23,30 antiserum identifies more than just B cells well with SmIg results.

suffkiently well with sheep erythrocytes (E) to allow their identification as T cells by the conventional method.3 Absolute numbers of E-rosetting lymphocytes were also decreased in AT patients (900 vs 1500-2500 cells/mm3). Figure lb compares B-cell levels in patients (27.%), parents (18.2%), and sib a This was demonstrated even more convincingly in an infant with severe combined immunodeficiency who had 9% E-rosetting lymphocytes but 55% T lymphocytes using the 3Al antiserum. Mitogen and allogeneic responses were extremely low in that patient.

505

ATAXIA-TELANGIECTASIA

lings (18.1%) to control values (l&l%), as measured by surface membrane immunoglobulin (SmIg) and by indirect immunofluorescence using a rabbit antiserum raised against human p23,30 (courtesy of J. Strominger and R. Humphreys), an antigen found on B lymphocytes, monocytes and a small percentage of T lympho cytes. Half of the patients had elevated B lymphocyte proportions; three had low levels; the mean level was significantly higher than control values (P < 0.01). One patient maintained such markedly elevated B-cell levels (absolute number = 720/mm3) that SmIg markers for kappa and lambda light chains were performed periodically to assess clonality. She had 28% kappa-bearing and 14% lambdabearing lymphocytes. Her B-cell levels have remained high for over 2 years with no apparent clinical counterpart. The anti-p23,30 indirect immunofluorescent staining tended to identify higher percentages of positive cells than did the SmIg markers (31.8% with anti-p23.30 versus 22.9% with polyvalent). We attribute this to Ia positivity on cells other than B lymphocytes, such as monocytes and T lymphocytes (11). a-Naphthyl acid esterase (ANAE) stains approximately 74.7 ? 4.3% of Enrosetting lymphocytes from normal donors and these lymphocytes are primarily, although not exclusively, T helper cells (4, 5, 12- 14). We found that five of ten patients had low values (Fig. 2). More than half of the parents and siblings also had low ANAE+ T lymphocytes. When the mean values for these groups were compared with normal data, they were each of marginal statistical significance (P = 0.01). (Follow-up studies with a battery of monoclonal T antisera are in progress.) EAC rosettes were normal in the AT patients (patient mean = 28.2% versus 29.8% for normal donors), but were slightly lower than normal in parents (25.8%, data not shown).

Normal Adult Range

AT

Parents

Sibs

FIG. 2. ANAE+/E+ cells are expressed as the percentage of E+ cells which contained the ANA.E “dot” on cytocentrifuged preparations. Note that patient, parent, and sibling groups had deficient levels of this T-cell subset.

506

GATT1 ET AL. . .

14Or 120 0 2 6 100 ” % 2 80

NOUlld Adult Range

b . t+ 60 5% . I L-----g

40

0

2 a

. .

20

AT

parents

sbs

controls (< 14 y/o)

FIG. 3. In virro lymphocyte responses to stimulation with PHA mitogen were deficient in about half of the patients. Parent and sibling groups were normal although wide variations were found.

PHA Responses

Responses to PHA were markedly depressed in 5 of 12 patients (Fig. 3). This did not correlate with length of illness or age. Responses of parents and siblings varied widely, but had normal mean responses. The depressed PHA responses were generally found in patients with depressed E-rosette levels, but discordant pairs were also noted. Although the P value for mean response of patients versus controls was significant (P < O.Ol), the PHA response alone was of little biologi-

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0

T

l

l

:

0 01

8

? 80 f0 0I 1 I

(0)

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I

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AT

Parents

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I Controls

FIG. 4. T suppressor cells were Con A-induced in patients, family members, and controls and their influence on normal mitogenic responses measured by thymidine uptake of normal cells. Although a few AT patients appeared to have decreased inducible T suppressor activity, wide variations in all groups, including controls, do not allow conclusive analysis without much additional data. The control points in parentheses represent mean values of repeated testing for two healthy donors which were outside our normal ranges. All other points represent results of a single testing.

507

ATAXIA-TELANGIECTASIA

cal or clinical relevance in evaluating individual cases because of the wide variations noted among patients. On the other hand, patients with abnormal PHA responses tended to maintain this pattern on repeated testing. Con A-Znduced

T Suppression

Figure 4 shows the data points for the four groups which were evaluated for T suppressor function. Conventional statistical analyses did not support differences between any of these four groups and further statistical manipulations were considered unwarranted without additional data. A possible trend toward lower T suppressor activity in PBL from AT patients was appreciated. However, repeated testing of two healthy donors (see parentheses in Fig. 4) showed low levels which fell outside two, and sometimes three, standard deviations from the mean, making it difficult to define normal ranges with confidence. Comparable multiple testing of patients was not feasible. Con A Capping

of Lymphocytes

When peripheral blood lymphocytes (PBL) were incubated in the presence of fluorosceinated Con A, a significantly greater percentage of lymphocytes from patients with AT had completed capping by 90 min at 37”C, as compared to adult controls (15% vs 7%; P < 0.001) or age-matched controls (15% vs 7.8%; P < 0.01) (Fig. 5). The mean value for the parents’ group was intermediate (11.1%; P > 0.01). Although one might expect intermediate values for a heterozygous popula-

AT

Parents

Sibs

Adult Controls

Agematched Controls

FIG. 5. Redistribution (capping) of FITC-labeling Con A after 90 min incubation of the cells at 37°C revealed markedly increased numbers of lymphocytes which had completed capping in the AT group. Mean for the parent group was intermediate between means for patient and control groups. No significant differences were noted between siblings, adult controls, and agematched controls. (For additional data, see Table 1.)

508

GATT1

ET AL.

TABLE

1

Con A CAPPING OF PBL AFTER 90 min INCUBATION (%) 4°C

37°C

Patient Group (N)

None

Ataxia-telangiectasia (12) Parents (13) Siblings (6) Adult controls (10) Age-matched controls (12)

37 42 34 51 44

Complete” 15 * 5.5 11 I 7 8

t ” + +

4.6 2.9 2.5 2.4

None 49 55 65 61 66

Complete” 5 3 3 2 2

t + t k 2

2.8 2.2 2.3 1.8 1.9

(1The percentage of cells with patching or incomplete (passive) capping can be obtained by subtracting (“none” + “complete”) from 100.

tion, this relationship was not clearly maintained when individual family members were compared (i.e., high levels in the patient group were not necessarily related to high or intermediate levels in the parent group). When capping conditions were altered to 4°C for 90 min, an increased percentage of capping of patients’ lymph* cytes was again noted but this was not statistically significant (Table 1). Capping values for normal children, age-matched for our younger patients, were not significantly different from those of adults (mean value for both groups combined was 7.4%). Capping results of patients and parents did not reflect changes in age, T- or B-cell levels, or PHA responsiveness, although such relationships might have been obscured by the relatively small numbers in our study. Neutrophil

Functions

The abnormal capping noted in patients’ lymphocytes prompted us to investigate membrane functions in other cell lineages. Neutrophil chemotaxis to the chemotactant, MLP, was decreased in all but three patients tested (Fig. 6). These three patients had been treated, several months before, with the immunopotentiator levamisole, but were not receiving any medication at the time of study. Parents’ n&trophils exhibited wide variations which ranged from clearly decreased to high normals. Random mobility of neutrophils across Millipore filters, thought to reflect the rigidity of the cell membrane itself, did not differ from normals to patients or parents. Cyclic Nucleotides As seen in Fig. 7a, CAMP levels in AT were found to be markedly increased in T lymphocytes of patients (15.7 pM/lO’ cells) as compared to the adult age-matched controls (4.1; P < 0.001) or the
509

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FIG. 6. Neutrophil chemotaxis toward a synthetic tripeptide, MLP, was significantly lower in the patient group (1.45), with little overlap of individual results. The mean for the parent group ( 1.g) was intermediate between means of patient and control (2.23) groups.

The difference between adult age-matched controls and the
(Zg) Levels

Serum IgA levels were decreased patients. IgM levels were elevated in elevated in 2 patients. IgG subclasses were measured in IgG2 levels (Fig. 9). Several patients

in 8 of 12 patients. IgG levels were low in 2 4 patients. IgE levels were low in 5 patients; 11 patients: all but 2 patients had decreased had low IgGl and IgG4 levels as well.

GATT1 ET AL.

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14 y/o controls

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FIG. 7. Cyclic nucleotide levels were measured on En-enriched lymphocyte preparations (~1% SmIg+) (a) Note markedly increased concentrations of CAMP in patients’ cells. Siblings also had elevated levels. CAMP concentrations in parents were normal, even when compared with age-matched controls. Controls under 14 years of age had higher levels than adults, as previously observed (6). (b) cGMP concentrations were elevated in patients’ cells. Parents and sibling group means were not significantly different from controls. Young controls had significantly lower concentrations than adult controls, as previously observed (6).

l

I

5

FIG. of 0.68 CAMP means

I

10 CAMP

I

15 lpM/107

/

I

I

20 cells1

25

30

8. Comparison of capping versus CAMP in lymphocytes of patients with AT. A correlation (r) was found. This correlation was also seen in the sibling group but not in the parent group whose levels were normal. The lowest CAMP value plotted could not be retested. Others represent of several measurements.

511

ATAXIA-TELANGIECTASIA

Normal Range

AT

parents

sibs

FIG. 9. Serum IgG, levels were below the normal ranges (age-corrected) in all but two AT patients. Details for normal ranges are given in Ref. (9).

Thymosin-a-1

Using a radioimmunoassay (7), thymosin-a-l serum levels were measured in 11 patients and family members. Both adult and child controls were tested simultaneously. We found no differences between the five groups (Fig. 10). Liver Studies

cx-Fetoprotein (AFP) was elevated in 9 of 12 patients. Patient J.R.‘s AFP level was normal; his mother’s level was very high. (Both mother and child were retested on three occasions.) In two siblings (R.G. and D.G.) with mental retardation and AT, the AFP levels were normal. Lactic acid dehydrogenase (LDH) was elevated in all but two patients, one of these two was J.R., who was 10 years old and in his fourth year since diagnosis. Alkaline phosphatase (AP) levels were elevated in J.R. and all other patients

I

AT

I

parents

I

sibs

I

adult controls

I

< 14 y/o controls

FIG. 10. Thymosin-a-1 levels as measured by RIA were not significantly different from controls in patients or family members. Cord blood serum was used as a daily control, however, no corrections of measured values were made.

512

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except one: T.R., a 20-year-old male with two affected siblings. T.R.‘s LDH was also normal, while his AFP was elevated (82 @ml). One of his affected siblings maintains a borderline AFP which was elevated at the time of the study. DISCUSSION

The major goals in the care and treatment of patients with ataxia-telangiectasia remain: (i) to understand enough of the underlying pathogenesis to allow a rational approach to therapy; (ii) to identify any heterogeneity within this group of patients; (iii) to identify a pathognomonic laboratory finding which would confirm early diagnoses; and (iv) to find a means of reliably identifying “carriers” or heterozygotes among the normal siblings of patients and within the general population. The latter goal may be of additional significance in identifying high-cancerrisk individuals, since there are data to suggest that the parents of AT patients may be at increased risk of developing cancer (15). Our investigations were focused primarily on the first goal, that of understanding the basic defect which leads to ataxia, to telangiectases, and to the myriad of abnormal laboratory findings. In this regard, three hypotheses have been commonly advanced: (i) an immunoregulatory defect (16- 19), (ii) a defect of DNA repair and/or replication (20, 21), and (iii) a hypophyseal defect (22). Our data certainly support the idea that immune functions of AT patients are typically and broadly perturbed, as would be expected for an immunoregulatory defect. Further, the Ig classes most commonly involved in serum deficiencies in these patients follow the order of the IGH genes on chromosome 14q, i.e., while CL, 6, 73, yl are expressed normally, ~2, ~4, E, and (Y are typically expressed as serum Ig deficiencies (23,41). On the other hand, we suspect that these various immunologic perturbations represent epiphenomena which, while they may prove of great importance in therapeutic manipulations, are not the common denominator of the disease process. The DNA repair defect is being addressed by us in separate studies and will be discussed elsewhere, The “hypophyseal defect” hypothesis was not supported by our finding normal thymosin-a-1 levels in all patients. However, our findings are contrary to previous reports (which we feel were based upon assays of lesser reliability) and further measurements of the various thymic factors are needed in AT patients. Since ablation of almost any endocrine organ leads to some immunologic consequences in animal models (24), a hypophyseal etiology for this disorder is still very plausible. We noted several abnormalities which relate to cell-surface changes and indirectly implicate the underlying cytoskeleton. In fact, most of the cellular abnormalities that have been reported in AT patients are compatible with the hypothesis of a cytoskeletal defect. Spindle formation of mitosis, cell motility, chemotaxis, capping, and neuronal growth and function all depend upon microfilaments and/or microtubular components of the cell scaffolding (25-27). We found an increase in the proportion of lymphocytes which completed cap ping in a standardized assay. While capping values in some patients exceeded those standard deviations from normal controls, other patients showed highnormal values. Even some parents showed this increased capping. Further evaluations of these findings are in progress. Enhanced Con A capping was previously

ATAXIA-TELANGIECTASIA

513

described in a patient with severe combined immunodeliciency and linked to microtubule disassembly (28, 29). Albertini er al. (30) suggest that microtubules limit the lateral movement and eventual capping of Con A receptors in normal cells and that it is the disassembly of these microtubules that allows capping to occur. Thus, it would appear that the enhanced capping observed in AT patients may reflect an inherent instability of microtubules. An abnormal energy metabo lism might provide the pathway to such microtubular instability, one that could certainly result from a single genetic defect. Neutrophil chemotaxis was decreased in most of our patients. Hill has described a role for cyclic nucleotides as modulators of leukocyte chemotaxis (31). Parker (32) suggests that CAMP is a positive effector of mitogenesis in lymphocytes. Conversely, Hadden and co-workers (33) propose a negative role for CAMP and a positive one for cGMP. Watson (34) argues that it is the rafio of CAMP/ cGMP that regulates lymphocyte reactivity. In lymphocytes from young animals, variation in cyclic nucleotides constitutes one of the earliest chemical events after activation by plant mitogens (35). In addition to finding increased CAMP levels in AT lymphocytes, we noted a correlation between CAMP levels and the percentage of lymphocytes completing Con A capping after 90 min. The possibility remains, however, that in our AT patients both the capping and the cyclic nucleotide abnormalities may reflect increased proportions of immature lymphocytes in the peripheral blood (29). This is being explored. Neurologic tissues contain high levels of cyclic nucleotidases while neuronal cells contain well-developed microtubular systems (25, 26). Sayhoun rt al. have recently described specific binding of adenylate cyclase to the cytoskeleton of erythrocytes (36). Puck has provided evidence for a similar relationship between CAMP and microtubules (37). Naeim, Walford, and coworkers have reported a relationship between decreased CAMP and capping in aged individuals (i.e., over 80 years) and in patients with Down’s syndrome (6, 38-40). Our data failed to provide any reliable means of identifying subsets of AT. While all members of one family had normal T-cell levels and two other families had normal capping percentages, a much larger experience would be needed to reliably document such familial patterns. Normal AFP levels are uncommon in AT and might be thought to identify familial AT subsets: however, in one of our families, one affected sibling maintains a borderline AFP level while two others have elevated levels. J. R., a l@year-old male with a normal AFP level (and a mother with an elevated level), does differ in some other laboratory parameters from the typical profile of AT patients in that his PHA is within normal range, his B-cell levels are normal, his capping was normal, his IgGz level was borderline low and his IgA level was normal. He could represent either a “forme fruste” or a subset of AT. On the other hand, he has the typical neurological and oculocutaneous signs of the disease and he continues to deteriorate neurologically despite few infections. In another family, two sisters with normal AFP and mental retardation could also represent an, as yet, unsubstantiated subset. However, both their immunologic parameters and their other clinical characteristics were quite similar to the rest of our patients. We have tried from our data to design an index which utilizes the weighted

NT’ NT * *

8

*

ANAE

*

CX-T

**

E

*

PHA

I;

*

T,

(’ Not all siblings were from the same family. Ir * denotes an abnormality of that parameter. ‘. Not tested.

1 2 -j 4 5 6

Donor

?

cap 7

*

* 8

CAMP

? *

*

cGMP

? 3

A/G ‘? * %

SmIg

*

LX-B

TABLE 2 MULTIPARAMETER ANALYSIS OF SIX AT SIBLINGS" &A B * * * * *

*

w2

AFP

w

I *

DNA

? 3

?

Heterozygote

::

2

2

P

ATAXIA-TELANGIECTASIA

515

averages of a group of parameters in combination to identify heterozygotes among the six siblings we have studied in depth. Table 2 summarizes these data. We found that IgA levels were borderline low in all six siblings tested. Thus far, we cannot reliably make an assignment of heterozygosity, even when we include the DNA repair data (to be published), since this index does not identify all parents (i.e., obligate heterozygotes) as heterozygotes. In conclusion, we anticipate that further analysis of the cytoskeletal integrity and intracellular biochemistry will prove revealing in understanding the basic etiology of the multiple anomalies seen in this disease. Recently developed genetic methodologies also allow, for the first time, that an inherited disorder, such as AT, be analyzed by first mapping the gene site (e.g., perhaps to 14q?), then decoding the genetic message and, thereby, pinpointing the source of the pathology. However, translating the defective genotype into its diseased phenotype will also be necessary in order to learn where and how to effectively treat these patients. Thus, all three levels of investigation, i.e., genetic, biochemical and immunological, appear to be important approaches to the treatment and cure of this fatal disorder. ACKNOWLEDGMENTS We thank Drs. Faramarz Naeim and Robert Sparkes for their helpful comments and discussions as this work was in progress. We thank Michael Boehnke for his assistance in the statistical analyses. We thank the families of our patients for their ongoing cooperation.

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