Genetic deficiency of the purine salvage enzyme adenosine deaminase (ADA) results in varying degrees of immunodeficiency, ranging from neonatal onset Severe Combined Immunodeficiency (SCID) to an adult onset immunodeficiency disorder. Multiple different mutations have now been identified in these immunodeficient patients. Additional mutations, initially identified in healthy individuals, abolish ADA in erythrocytes but retain 10-80% of activity in non-erythroid cells (`partial deficiency mutations'). In general, severity of disease correlates inversely with the amount of residual ADA expressed by the mutant enzymes and directly with the accumulation of the toxic metabolites deoxyATP and deoxyadenosine. We report two newly identified mutations (Y97C and L106V), both carried on the same allele of an immunodeficient patient who was diagnosed prenatally and successfully transplanted with haploidentical bone marrow. Based on the ability of mutant cDNAs to express ADA in vitro, the L106V mutation resulted in activity similar to `partial' mutations (30% of normal) while the Y97C mutation resulted in detectable but markedly reduced activity (1.5% of normal). However, the presence of both mutations on the same allele virtually abolished detectable enzyme activity. Analysis of the crystallographic structure of ADA to understand the marked deleterious effect of the Y97C mutation suggested a previously unappreciated role of salt bridges in the catalytic mechanism of ADA. The patient was also heteroallelic for a previously described deletion of the promoter and exon 1. Testing of additional patients in whom we had not identified a mutation on the second allele revealed presence of this deletion in three of four patients tested. This deletion is therefore relatively common, accounting for 10% of almost 100 chromosomes studied by this and other laboratories, but is easily missed by currently used methods of mutation detection. Lastly, the finding of two mutations on the same allele that interact to reduce residual enzyme function emphasizes hazards in evaluating potential genotype-phenotype correlations in individuals analyzed only for the presence of single specific mutations.
Adenosine deaminase (ADA) is an enzyme of the purine salvage pathway that catalyzes the deamination of adenosine and 2' deoxyadenosine to inosine and 2' deoxyinosine, respectively. Inherited deficiency of ADA results in an autosomal recessive immunodeficiency disorder of varying severity. The phenotypes include classical neonatal onset rapidly fatal Severe Combined Immunodeficiency (SCID) with absence of both cellular and humoral immunity; a delayed onset, more slowly progressive disease characterized by initial retention of some humoral immunity; a later juvenile onset disease often not diagnosed till 5-8 years of age, with progressive attrition of immune function and a recently described adult onset disorder. Additionally, children have been found by population screening who have no apparent immune deficit and who lack enzyme activity in erythrocytes but express variable residual ADA (5-80% of normal ADA) in other cell types (`partial' ADA deficiency). In general, the degree of enzyme deficiency, accumulation of the toxic metabolite deoxyATP and urinary excretion of the substrate deoxyadenosine grossly correlate with severity of disease. Consistent with this diversity, >40 mutations spanning much of the 32 kb, 12 exon ADA gene on chromosome 20q13 have been identified, including deletions, missense, nonsense and splicing mutations (1 -5 ).
We have molecularly and biochemically studied the sib of a patient who died with ADA-SCID. The sib was diagnosed prenatally as ADA deficient, the diagnosis confirmed at birth and the child successfully transplanted with haploidentical bone marrow. We newly identified two missense mutations (Y97C and L106V) on the same allele that interacted to abolish residual ADA activity. The second allele contained a previously described deletion of the promoter and exon 1 (6 -8 ), which, we now report, is a relatively frequent mutation.
Prenatal testing for ADA deficiency by assay of ADA in cultured amniotic cells, performed because of the death of a prior sib with ADA-SCID, revealed the presence of an affected fetus (0.07 versus normal = 7.5 ± 4.1 nmol/mg protein/min). The prenatal diagnosis was confirmed after birth by demonstration of <1% of normal ADA activity in the child's RBCs and peripheral blood mononuclear cells (Table 1 ). DeoxyATP content of erythrocytes was markedly elevated (Table 2 ), similar to concentrations found in patients with SCID but not as high as those we have found in patients with neonatal onset of disease. Excretion of deoxyadenosine in urine was similarly elevated.
Table 1 . ADA enzyme activity in erythrocytes (RBC) and peripheral blood mononuclear cells (PBM)
Table 2 .
Digestion of DNA from amniotic cells with EcoRI or HindIII and hybridization of Southern blots with a full length ADA cDNA suggested relatively lesser amounts of DNA in restriction fragments containing exon 1 as compared with fragments containing other exons (not shown). Based on these observations, we first sought to determine if one allele of the patient carried a previously reported deletion of 3.73 kb encompassing the promoter and exon 1 that results from recombination between repetitive Alu sequences (6 ,7 ). PCR amplifications using primers flanking the previously reported deletion generated a 481 bp fragment in the patient and her father and in DNA from an individual homozygous for this deletion (not shown) but not in the mother or in normal (Fig. 1 ). The sequence at the recombination joint was identical to that previously reported in two patients (6 ,7 ) but differed from that reported in a third patient (8 ). Additionally, we tested DNA from four other patients in whom we had been able to identify a mutation on only one of the alleles. The deletion of the promoter and exon 1 was identified in three of these additional patients (Lco, GM02436 and Fa). All of the patients carried the same, more frequent, recombination joint (not shown).
To evaluate the ability of the Y97C/L106V mutations and of each of the mutations individually to express ADA, each of the mutations alone and both mutations together were introduced into a normal ADA cDNA. The mutant cDNAs were transiently expressed by co-transfection with a [beta]-galactosidase([beta]-gal) cDNA into Cos cells. Lysates were adjusted to contain known amounts of [beta]-gal activity (both to correct for any differences in efficiency of transfection and to allow for semi-quantitative analysis), electrophoresed in starch gels and ADA activity assessed by in situ staining. Activity was compared with that expressed by a normal ADA cDNA and by different mutant cDNAs (R156H, L107P, R211C, T233I or A215T) (9 ,14 ,32 ), using different amounts of total [beta]-gal activity to allow for semi-quantitative analysis as illustrated in Figure 3 and summarized in Table 3 . The L106V mutation expressed considerable ADA, although substantially less than the normal ADA cDNA (30% of normal) (Fig. 3 , Table 3 ). Comparison of the L106V mutation with several previously described partial mutations, revealed that the L106V mutant expressed the greatest activity among the four mutations tested, with L106V (~30%) > T233I (~16-20%) > A215T (~8%) > Arg211Cys (~4%) (Table 3 and data not shown). By contrast to the L106V mutant cDNA, the Y97C mutation expressed only very low amounts of ADA activity (1-1.5% of normal). Most significantly, presence of both mutations together (L106V/Y97C), virtually abolished expression of ADA activity (Fig. 3 , Table 3 ). Lastly we compared the activity of the L106V mutant with a mutation (L107P) at the adjacent codon (14 ). In contrast to the L106V mutant, the L107P mutant cDNA did not express detectable activity (not shown).
We have studied a child diagnosed prenatally as ADA deficient, confirmed by laboratory tests after birth as immunodeficient and ADA deficient, and successfully transplanted with haploidentical marrow prior to development of the clinical complications of immunodeficiency. Based on the history of her affected sib, who retained immunoglobulins and survived to 27 months of age, the phenotype would fall into the category of `delayed', rather than `fulminant neonatal' onset. The metabolite concentrations present in this second child (not determined in the proband) were consistent with the phenotype of the proband. Molecular studies of the child and her parents as well as of additional patients led to (i) unexpected discovery of a complex allele, (ii) demonstration of the frequent lack of diagnosis of a relatively frequent mutation by currently used PCR based diagnosis, and (iii) the suggestion of a previously unappreciated role of salt bridges in the catalytic mechanism.
ADA activity, deoxyATP and deoxyadenosine concentrations were determined essentially as previously described (26 ). ADA was determined in the patient's samples by measuring conversion of radiolabeled adenosine to inosine and hypoxanthine in the presence and absence of the specific inhibitor EHNA (27 ). RBC ADA of parents and normal was determined using a previously described spectrophotometric assay (27 ).
Exons 1, 2, 3, 4-5, 6, 7-9 and 10-11 were amplified from genomic DNA as previously described (19 ,28 ,29 ) (Table 5 ) and sequenced using cycle sequencing with a fMole kit (Promega) or a thermo sequenase radiolabeled terminator cycle sequencing kit (Amersham). A deletion of the promoter and exon 1 was identified by PCR of genomic DNA using primers flanking the Alu repeats involved in generation of the deletion: sense, -1641 to -1611 nt (BG4850), 5'-GATGAAATGAAATGAAGCTACCATCCACCCC-3' and antisense, 2090 to 2060 nt (BG4849), 5'-CTTGATGGGCTGTTACAGAAGAACTGCAAGG-3' (numbering as in ref. 30 ).
Table 5 .
Mutations were introduced into the normal ADA cDNA in the expression vector pSV2 (pSV2ADANL) by primer directed mutagenesis as described by Ho et al. (31 ), as we have previously described (32 ). In brief, using PSV2ADANL as template, PCR amplification was used in a two step procedure to generate a fragment that contained the newly introduced mutation surrounded by two unique endogenous restriction sites. Following digestion and purification, the fragment containing the mutation was exchanged by ligation for the homologous fragment in pSV2ADANL. Specifically, two flanking primers, 5' to a unique SfiI site in the pSV2 vector (nt 57) and 3' to a unique PpumI site in the ADA cDNA, and sense and antisense mutant primers specific for a Y97C or a L106V mutation were used for amplifications. For generation of cDNA containing both mutations, we first introduced the Y97C mutation which was then used as template for introduction of the L106V mutation. All cDNAs were released from pSV2 by HindIII digestion and cloned with the correct orientation in the HindIII site of pcDNA3 (Invitrogen). All clones were analyzed for sequence using cycle sequencing with either a fMole kit (Promega) or a thermo sequenase radiolabeled terminator cycle sequencing kit (Amersham) to confirm presence of the mutations and absence of PCR errors.
Plasmids were amplified and DNA purified using a Qiagen Plasmid Maxi Kit. A 100 mm plate of Cos cells was co-transfected with 20 µg of an ADA construct and 1 µg of pRSVZ ([beta]-galactosidase gene driven by an RSV promoter) by calcium phosphate-DNA co-precipitation essentially by standard methods with minor modifications as previously described (32 ). Medium was changed at 8 h, cells were harvested by scraping at 48 h, washed pellets resuspended in 50 µl 0.001 M Na-phosphate buffer (pH 7.0), and lysed by five cycles of freezing and thawing using dry ice/ethanol and a 37°C water bath and sonication for 10 s at 50 W. The supernatant was collected after centrifugation for 15 min at 4°C in an Eppendorf microfuge.
[beta]-Galactosidase activity was determined in the lysates of transfected cells as previously described (32 ). Lysates containing known amounts of [beta]-galactosidase activity (to control for efficiency of transfection) were electrophoresed in starch gels to separate human from endogenous Cos ADA. Gels were stained to detect ADA activity as previously described (32 ), using incubation at 37°C for 1-2 h.
Coupled transcription and translation was performed using a TNT Coupled Reticulocyte Lysate kit (Promega) either with incorporation of [35S]methionine (NEN Dupont; >1000 Ci/mmol) or unlabeled. Labeled protein was analyzed on discontinuous SDS- polyacrylamide 10% gels with autoradiography. Unlabeled protein was analyzed for ADA activity on starch gels as described (32 ).
ADA- imunodeficiency was diagnosed in the proband at age 7 months, following pneumocystis pneumonia. As previously reported in detail (33 , pt#115), prior to haploidentical transplant with paternal marrow at age 27 months, he was markedly lymphopenic with reduced CD4+ and CD8+ cells. He showed minimal responses to mitogens and alloantigens and no response to candida, streptokinase/streptodornase or poliomyelitis antigens. Immunoglobulins were within normal ranges (IgG = 414, IgA = 45 and IgM = 20 mg/dl versus age matched normal of 762 ± 209, 50 ± 24 and 58 ± 23, respectively), but no specific antibodies were found to tetanus, diphtheria toxoid or E.coli. He died 9 days after the start of chemoablation, due to cyclophosphamide induced myocardopathy.
The patient studied here is a sib of the deceased proband and was diagnosed prenatally as ADA deficient. In accord with the parents' wishes, the pregnancy was carried to term and immunodeficiency as well as enzyme deficiency confirmed postnatally. She was markedly lymphopenic with markedly reduced CD4+ T cells (2%). Her lymphocytes were unresponsive to stimulation in vitro by mitogens, alloantigens and antigens. The patient received a haploidentical transplant at 8 weeks of age following preparation at the same dosages as her brother (33 ). Four months after transplant she had vigorous in vitro proliferative responses to mitogens and alloantigens and 1 year after transplant, vigorous antigen responses. All immunoglobulin values were low for the first 2 years after transplant, with IgA not detected for the first 6 months, although specific antibody responses to tetanus, diptheria toxoids and E.coli were detected in normal titers. By 3 years after the transplant, the immunoglobulin levels were within the normal ranges. Her tests of immunity have remained normal and she has lived a normal life now over 9 years since transplant without undue susceptibility to infection and without gamma globulin therapy or antibiotic prophylaxis.
We thank Dr K. Hirschhorn for the critical reading of this manuscript. This work was supported by a grant from the NIH AI R2910343 to R. Hirschhorn.
Human Molecular Genetics
Pages
Introduction
Results
Biochemical studies
Molecular analysis of mutations
In vitro studies of expression by the two missense mutations found on one allele of the patient
Discussion
Materials And Methods
Determination of ADA activity and metabolite concentrations
Identification of mutations
Introduction of mutations into the normal ADA cDNA
Transfection into Cos cells and transient expression of ADA by normal and mutant clones
Determination of [beta]-galactosidase activity and in situ detection of ADA activity
In vitro transcription-translation
Case history
Acknowledgements
References
RBCsa
PBMb
Genotype
Patient
0.025
0.01
Y97C&L106V/del. exon 1
Mother
41
NT
Y97C&L106V/Normal
Father
39
NT
Normal/del. exon 1
ADA-SCIDsc
<0.50
9.4 (0.4-25)
Normals
83.8 (58-134)
1190.0 ± 128
dATPa RBCs
(range)
dArb Urine
(range)
Patient
687
442
SCID (n = 9)c
1160
(327-2248)
1033.0
(442-2500)
CID (n = 2)d
210
(174-247)
170.0
(54-270)
Partial' (n = 11)e
16
(4-38)
6.0
(2-13)
Normal (n = 38)
4
(1-14)
<0.2
<0.2
Exon 1
5' sense primer (H063A): 5'-cacgaattcACCGAGCCGGCAGAGACCCAC
3' antisense primer (H019): 5'-ACTTGACAGACAGCGAAACTGAGACCCAGA
Exon 2
5' sense primer (MSH029): 5'-GGAGCACAAGCTTTGGAATTGGGCTTGGGTTA
3' antisense primer (MSH031): 5'-ACACCAGGAGGACAAGACTCAGAGGCCCAGAA
Exon 3
5' sense primer (BG4299): 5'-cgagaattcGTTCTCAGTTTCCCCATCTGTCCAGTGGGAGCAG
3' antisense primer (BG4300): 3'-aggaagcttCTGAGGGACAGGCCTGGTCCTAGTCATAGGGAT
Exon 4-5
5' sense primer (BG10474): 5'-ATGAAGTGGGTTTAATCTGCCAAGGGTTGGGGATG
3' antisense primer (DG747): 5'-TCATGAAGCCCGAAGTTCATGCCAGTGGGCTCAAG
Exon 6
5' sense primer (BG3419):5'-CATAGCAGTTAGGATTTGAAGACACTGAGCCC
3' antisene primer (BG3418): 5'-AGGAGACACCATGGTCCCTGGTTCTTGTGAT
Exon 7-9
5' sense primer (KH060):5'-ATGCTGTTGAAGCAGGCAGCATGACTAGGA
3' antisense primer (KH061): 5'-TGCCTGCTTCCCAGGGTGTCGAAGAGATTT
Exon 10-11
5' sense primer (BG2714):5'-AGGCTGCTGTGAGGATCAAAGGCGGGTGAA
3' antisense primer (BG2715): 5'-TGCTAGAAGTCCCACAGAAAGCCACACTGG
Exon 12
5' sense primer (BG4528): 5'-TGGCAAGGCATCTTCTTGCCATCATAGGAAGG
3' antisense primer (BG4527): 5'-CTCTTGGCCCACAGCATTGGGATCTCTGG
REFERENCES
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Oxford University Press, 1997