Importance of the determination of enzymatic activity in the diagnosis of deficiency of adenosine deaminase 2 (DADA2)

Abstract

Objective

To describe the usefulness of determining the enzymatic activity of adenosine deaminase 2 (ADA2) in patients with suspected ADA2 deficiency (DADA2).

Method

Retrospective multicenter study. Review with analysis of the clinical, biochemical and genetic data of the patients in whom the enzymatic activity of ADA2 has been determined by spectrophotometric method.

Result

In 3 of the 20 patients, the diagnosis of DADA2 was confirmed by the combination of reduced enzyme activity and biallelic pathogenic variants in the CECR1 gene. In 2 patients with variants of uncertain significance in CECR1, the study of enzymatic activity allowed to rule out the disease.

Conclusions

The reduced enzymatic detection of ADA2 confirms the diagnosis of DADA2, particularly important in carriers of variants of uncertain significance in CECR1.

Resumen

Objetivo

Describir la utilidad de la determinación de la actividad enzimática de adenosina desaminasa 2 (ADA2) en los pacientes con sospecha de déficit de ADA2 (DADA2).

Método

Estudio retrospectivo multicéntrico con análisis de los datos clínicos, bioquímicos y genéticos de los pacientes a los que se ha determinado la actividad enzimática de ADA2 mediante método espectrofotométrico.

Resultado

En tres de los 20 pacientes se confirmó el diagnóstico de DADA2 mediante la combinación de actividad enzimática reducida y variantes patogénicas bialélicas en el gen CECR1. En dos pacientes portadores de variantes de significado incierto en CECR1, el estudio de actividad enzimática permitió descartar la enfermedad.

Conclusiones

La actividad enzimática reducida de ADA2 confirma el diagnóstico de DADA2, de especial importancia en los portadores de variantes de significado incierto en CECR1.

Introduction

Deficiency of adenosine deaminase 2 (DADA2) is a monogenic autoinflammatory disease inherited in an autosomal recessive manner caused by loss-of-function mutations in the CECR1 gene (cat eye syndrome chromosome region, candidate 1), also called ADA2 gene. This gene, located on chromosome 22q11.1, codes for the enzyme adenosine deaminase 2 (ADA2)^1,2, which promotes the differentiation of monocytes to macrophages and stimulates the development of vascular endothelial and haematopoietic cells.

The presence of symptoms is a consequence of the absence of enzyme activity and, to date, only clinical manifestations have been described in patients with two mutated alleles, in most cases as compound heterozygosity. At a biochemical level, the presence of two mutated alleles translates into the absence of enzyme activity, while the presence of a single mutation is related to 50% enzyme activity, with no disease association.

Patients with DADA2 experience a multisystemic proinflammatory state with impaired vascular endothelial integrity associated with a mild immunodeficiency component, more evident in B cells³. Since the initial description of the disease in 2014, the phenotype has broadened, accounting for highly variable clinical features, making the identification of patients difficult. Four main phenotypes are distinguished: neurological, vascular disease, haematological abnormalities and immunodeficiencies. With the availability of current laboratory techniques, the number of patients diagnosed with DADA2 continues to increase, finding patients with symptoms as diverse as any systemic vasculitis⁴.

The diagnosis of DADA2 is made by demonstrating the presence of a compatible genotype and/or a significant reduction in ADA2 enzyme activity. ADA2 enzyme activity determination is only carried out in some centres, but it is of great help in assessing patients with suspected DADA2 by assisting in the interpretation of genetic studies in which variants of uncertain significance (VUS) or pathogenic variants on a single allele are detected.

Treatment with anti-TNF-alpha drugs has been shown to be effective in preventing new ischemic events^5,6, therefore, it is recommended to rule out this disease in patients with early-onset stroke, recurrent stroke and/or those in whom elevated acute-phase reactants coexist at the time of the ischemic event. Due to the underlying endothelial defect, these patients are at higher risk of haemorrhagic transformation than patients with stroke of other origin, so the initiation of antiplatelet and/or anticoagulant drugs is not indicated initially.

The aim of this paper is to review our series of patients with enzymatic determination of ADA2 and to describe its utility.

Patients and methods

Multicentre retrospective study of patients with ADA2 enzyme activity determination, requested according to the inclusion criteria, between October 2018 and November 2020. Three health centres have participated in the study. One family included in the study came from the Complejo Hospitalario de Navarra and the other from the Hospital Mútua de Terrassa, and were referred to Hospital Sant Joan de Déu, as it is a reference centre for autoinflammatory diseases, following the recommendations of Single Hub and Access point for paediatric Rheumatology in Europe (SHARE) for the follow-up of this type of patients in specialised centres with multidisciplinary care.

The criteria for the determination of ADA2 enzyme activity were: (i) presence of phenotype compatible with DADA2, (ii) detection of VUS in CECR1, and/or (iii) extension study to relatives of patients with DADA2.

The determination of enzyme activity was carried out in the biochemistry laboratory of the Sant Joan de Déu Hospital using a spectrophotometric method adapted for the determination of ADA2 based on previously described methods^7,8. The assay monitors the rate of release of ammonia (NH₃) from adenosine by coupling to the glutamic dehydrogenase (GDH)-catalysed reaction of α-ketoglutarate in the presence of nicotinamide adenine dinucleotide (NADH). Total ADA activity is determined by incubating the samples at 37 °C and measuring the absorbance at 340 nm at 30 and 90 min in a spectrophotometer (Jasco V-530). In parallel, the activity of ADA2 is measured by performing the same procedure incubating with EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), an enzyme that inhibits ADA1 but not ADA2, allowing measurement of the decrease in NADH produced only by the ADA2 enzyme. All reagents used were from Merck KGaA (Darmstadt, Germany).

Results

Twenty patients met the inclusion criteria. Table 1 summarizes their demographic data, enzyme activity levels, and genotype.

Table 1.

Demographic data, reason for enzyme determination and age at the time of screening, laboratory parameters including enzyme activity levels, variants detected in the CECR1 gene of the study patients and final diagnosis.

Patient	Sex	Reason for requesting the enzyme study	Age at disease diagnosis	Age at enzyme determination	White blood cells	Neutrophils	Erythrocyte sedimentation rate	C-reactive protein	Immunoglobulin G	Immunoglobulin A	Immunoglobulin M	Enzyme activity level	CECR1 gene variants	Final diagnosis
			[years]	[years]	[10^3/µL]	[10^3/µL]	[mm/1st hour]	[mg/L]	[mg/L]	[mg/L]	[mg/L]
1	Female	AS	0.5	4	5.4	2.4	25	4.1	291	13	45	14.3	WT/WT	Immunology study pending completion
2	Female	AS	8	9	7.3	2.0	37	16.5	1641	2160	560	2.2	V240M/P251L	DADA2
3	Female	AS	6	11	9.2	5.7	2	11.8	5893	603	636	10.10	Not performed	Primary immunodeficiency with defect in TLR3
4	Female	Cutaneous PAN	8	14	7.0	4.5	3	1.0	11,132	2667	3890	0.9	G47R/G47R	DADA2
5	Female	Macular lymphocytic arteritis	11	11	7.5	3.1	2	0.2	13,401	1514	1382	14.9	Not performed	Macular lymphocytic arteritis
6	Male	Cutaneous PAN	10	11	1.2	1.6	7	6.2	14,822	1184	1083	13.8	Not performed	Cutaneous PAN
7	Female	Leukocytoclastic vasculitis	16	16	8.2	5.3	5	0.3	9319	1363	1316	19.7	WT/WT	Sjogren's syndrome
8	Female	Livedo reticularis and fever	8	12	5.2	2.7	2	0.2	11,055	1258	1006	13.2	WT/WT	Undefined autoinflammatory disease
9	Male	Systemic PAN	2	6	15.3	10.9	29	102.4	1336	224	227	11.9	WT/WT	Systemic PAN
10	Female	Systemic PAN	11	11	8.2	5.8	43	42.2	12,178	1162	892	8.7	WT/WT	Systemic PAN
11	Female	Systemic PAN	16	16	10.9	7.7	50	123.7	17,864	5037	1210	16	WT/WT	Systemic PAN
12	Female	VUS in CECR1	2	2	15.1	4.6	32	4.8	10,766	501	1455	7.3	c.973-2A >G heterozygosity	RF-positive polyarticular JIA
13	Female	VUS in CECR1	3	14	9.2	7.1	16	78.8	1281	154	109	17.2	R49W/WT	Undefined autoinflammatory disease
14	Female	Familial study	N/A	37	7.0	4.5	12	3.4	11,741	4241	374	8.3	G47R/WT	N/A
15	Male	Familial study	N/A	57	13.4	9.3	4	4.2	8461	2515	344	5.8	G47R/WT	N/A
16	Female	Familial study	N/A	48	N/A	N/A	N/A	N/A	N/A	N/A	N/A	6.7	P251L/WT	N/A
17	Male	Familial study	N/A	52	N/A	N/A	N/A	N/A	N/A	N/A	N/A	8.5	V240M/WT	N/A
18	Female	Familial study	N/A	26	6.3	3.8	2	1.1	N/A	N/A	N/A	6.6	P251L/WT	N/A
19	Male	Familial study	N/A	29	8.1	4.0	2	0.5	N/A	N/A	N/A	5.3	P251L/WT	N/A
20	Male	Familial study	N/A	23	4.2	2.9	2	3.7	4007	372	85	1.7	V240M/P251L	DADA2

AS: Acute stroke; JIA: juvenile idiopathic arthritis; RF: rheumatoid factor; N/A: not applicable; PAN: polyarteritis nodosa; VUS: variant of uncertain significance; WT: wildtype.

Normal values: White blood cells 4500–13,000/µL; neutrophils 1800–8000/µL; erythrocyte sedimentation rate: <15 mm/1st hour; C-reactive protein: <15 mg/L; immunoglobulin G: 6500–15,000 mg/L; immunoglobulin A: 760–3900 mg/L; immunoglobulin M: 400–3450 mg/L; ADA2: by age: from 0 to 17 years: 5.08–23.76 U/L; >18 years: 8.48–17.52 U/L.

The determination of enzyme activity was performed in 13 patients on clinical suspicion. The genetic study identified a VUS in CECR1 in two of these 13 patients, and it was the enzyme activity that allowed ruling out the presence of disease. The diagnosis of DADA2 was confirmed in three of the 20 patients by a combination of reduced enzyme activity and compatible genotype (patients 2, 4 and 20).

One of the first-degree relatives studied in the context of intra-familial segregation of the variants detected in the two index cases of both families (patient 20) carried the same pathogenic variants as his sister (patient 2).

Discussion

DADA2 is a newly described disease in which virtually any organ can be affected, acute phase reactants are often elevated and patients with a “vascular accident” phenotype often have previous accident lesions on neuroimaging⁴. Although most patients have a paediatric onset, adult-onset cases have been reported, so age should not be a reason for exclusion of the disease⁹.

At present, given the lack of access to the determination of ADA2 activity, most centres base the diagnosis of DADA2 on genetic testing.

Recent publications have demonstrated the presence of disease in patients initially identified as carriers, where virtually undetectable enzyme activity prompted the completion of the genetic study with more advanced techniques that allowed the identification of hidden variants in CECR1¹⁰ and the diagnosis of patients, with consequences for treatment and prognosis.

Once our own reference values for ADA2 activity have been established for our population, it has been found that patients with DADA2 have a practically undetectable enzyme activity, clearly distinguishable from healthy controls and heterozygous patients; for this reason, the determination of ADA2 enzyme activity would be useful even before genetic testing is carried out. Furthermore, as it is a recently described disease, the identification of VUS is common, and the enzyme activity is essential in the interpretation of its pathogenicity.

Given the implications of the diagnosis of DADA2, it is recommended to rule out this disease in the presence of any compatible phenotype. Once diagnosed, it is recommended to start anti-TNF-alpha treatment and to offer screening to relatives, as first-degree relatives have been diagnosed in the pre-symptomatic phase⁹.

In our case, once the disease was detected in the two index cases of both families, a further intra-familial segregation study of the variants was carried out. Through this genetic study, only one family member was found to be affected, previously classified as systemic panarteritis nodosa (PAN), which made it possible to establish the diagnosis of DADA2 and initiate treatment with anti-TNF-alpha. The rest of the relatives studied carried only one pathogenic variant, which coincided with their levels of enzyme activity in the heterozygous range.

Although anti-TNF-alpha treatment does not correct the enzyme defect, it has demonstrated efficacy in controlling most of the clinical manifestations (ischaemic manifestations, vasculitis, hepatomegaly…). Since the phenotype can change with the course of the disease, even within the same family¹⁰, it is recommended to consider anti-TNF-alpha treatment in all patients, regardless of their phenotype, as primary prevention of stroke.

In our series of patients, the determination of enzyme activity has allowed (i) confirming the diagnosis of DADA2 in two patients with compatible genotype, (ii) identifying the presence of disease in a first-degree relative, establishing a monogenic cause of systemic PAN and motivating a change in immunosuppressive treatment, and (iii) ruling out DADA2 in two patients with inflammatory disease who were VUS carriers in CECR1.

Conclusions

Reduced or undetectable enzymatic determination of ADA2 confirms the diagnosis of DADA2, of particular importance in carriers of VUS in CECR1 and in cases with stroke phenotype in which it is recommended to avoid antiplatelet and/or anticoagulant drugs, given the high risk of haemorrhagic transformation.

Funding

There has been no funding of any kind for the development of the study.

Conflict of interests

The authors declare no conflict of interest.