Electroclinical features and phenotypic differences in adenylosuccinate lyase deficiency: Long‐term follow‐up of seven patients from four families and appraisal of the literature

Abstract Objective Adenylosuccinate lyase (ADSL) deficiency is a rare inherited metabolic disorder with a wide phenotypic presentation, classically grouped into three types (neonatal, type I, and type II). We aim to better delineate the pathological spectrum, focusing on the electroclinical characteristics and phenotypic differences of patients with ADSL deficiency. Patients and Methods Seven patients, from four different families, underwent serial electroencephalogram (EEG), clinical assessment, and neuroimaging. We also performed a systematic review of the cases published in the literature, summarizing the available clinical, neurophysiological, and genetic data. Results We report seven previously unreported ADSL deficiency patients with long‐term follow‐up (10–34 years). From the literature review, we collected 81 previously reported cases. Of the included patient population, 58 % (51/88) were classified as having ADSL deficiency type I, 28% (25/88) as having type II, and 14% (12/88) as having neonatal. The most frequently reported pathogenic variants are p.R426H homozygous (19 patients), p.Y114H in compound heterozygosity (13 patients), and p.D430N homozygous (6 patients). In the majority (89.2%), disease onset was within the first year of life. Epilepsy is present in 81.8% of the patients, with polymorphic and often intractable seizures. EEG features seem to display common patterns and developmental trajectories: (i) poor general background organization with theta‐delta activity; (ii) hypsarrhythmia with spasms, usually adrenocorticotropic hormone‐responsive; (iii) generalized epileptic discharges with frontal or frontal temporal predominance; and (iv) epileptic discharge activation in sleep with an altered sleep structure. Imaging features present consistent findings of cerebral atrophy with frontal predominance, cerebellar atrophy, and white matter abnormalities among the three types. Significance ADSL deficiency presents variable phenotypic expression, whose severity could be partially attributed to residual activity of the mutant protein. Although a precise phenotype‐genotype correlation was not yet feasible, we delineated a common pattern of clinical, neuroradiological, and neurophysiological features.


| INTRODUCTION
Adenylosuccinate lyase (ADSL) deficiency (OMIM 103050) is a rare autosomal recessive defect of the purine biosynthetic pathway caused by mutations in the ADSL gene, located on chromosome 22q13.1-q13.2. 1 From the first cases described by Jaeken and Van Den Berghe, 2 currently, close to a 100 cases can be retrieved from the literature.ADSL is an enzyme that plays a key role in purine metabolism, catalyzing two non-sequential steps: first, the conversion of succinylaminoimidazole carboxamide ribotide (SAICAr) to aminoimidazole carbozamide ribotide, and second, the conversion of adenylosuccinate (S-AMP) to adenosine monophosphate (AMP).ADSL deficiency causes the buildup of by-products, that is, SAICAr and S-ADO, detectable in patients' CSF, urine, and plasma.The clinical presentation associated with ADSL deficiency can be extremely heterogeneous and, based on the age of onset and clinical severity, can be broadly classified into three main groups 1 : • Type I or severe: most reported cases fall into this category.The onset is generally within the first months of life, and affected patients display the whole symptomatic spectrum of the disease, that is, severe developmental delay, epilepsy with intractable seizures, marked autistic features, axial hypotonia with limb hypertonia, dystonia, and ataxia. 3 Type II or mild: the onset of symptoms is generally within the first few years of life, but they can present later in life.Patients with type II ADSL deficiency generally present with mild to moderate developmental delay, autistic features, and variable pyramidal and extrapyramidal signs.They are less often characterized by seizures compared to the other groups. 4 Neonatal (N): The most severe form that presents at birth.Affected neonates are characterized by impaired intrauterine growth, microcephaly, fetal hypokinesia, a lack of heart rate variability, severe muscular hypotonia often leading to mechanical ventilation, resistant seizures, and early death. 5ilepsy in ADSL deficiency is present in a consistent portion of the affected individuals. 1Seizures tend to display wide semiological variability and are often intractable, severely affecting the quality of life and the outcome of these patients. 6Here we describe seven previously unreported cases from four different families.We also performed a review of ADSL cases, attempting to establish possible genotype-phenotype correlations and highlighting common clinical, neuroradiological, and neurophysiological features with specific reference to their epileptic background.

| METHODS
We collected patients with a diagnosis of ADSL deficiency in two Italian centers ("Vittore Buzzi Children Hospital" in Milan and "ASST Spedali Civili" in Brescia) and one center in France (University Hospitals of Lyon).Clinical present consistent findings of cerebral atrophy with frontal predominance, cerebellar atrophy, and white matter abnormalities among the three types.
Significance: ADSL deficiency presents variable phenotypic expression, whose severity could be partially attributed to residual activity of the mutant protein.
Although a precise phenotype-genotype correlation was not yet feasible, we delineated a common pattern of clinical, neuroradiological, and neurophysiological features.

K E Y W O R D S
ADSL deficiency, EEG patterns, epilepsy, genotype-phenotype correlation, monogenic diseases

Key Points
• Adenylosuccinate lyase deficiency is a rare inherited metabolic disorder with a wide phenotypic presentation.
• Epilepsy is a core feature of the disease, with polymorphic and often drug-resistant seizures.
• Patients display common electroencephalogram features and evolutionary trajectories.
• The severity of the phenotype seems to correlate with mutant protein residual activity.
data were retrieved retrospectively from clinical registries and prospectively through interviews with patients, their families, and/or caregivers.Electroencephalographic (EEG) and video-EEG recordings were available for all the patients at epilepsy onset and follow-up.EEGs were obtained by a digital acquisition system, placing scalp electrodes according to the international 10-20 system.Selected patients underwent additional neurophysiologic investigations (ie, visual, auditory, and somatosensory evoked potentials and electroretinograms).All patients underwent sequential brain magnetic resonance (MRI), and the images were reviewed and discussed with a trained pediatric neuroradiologist.Five patients (Pt.3-Pt.7) underwent urine SAICAr and S-Ado testing.All patients and their parents underwent genetic analysis using genomic DNA extracted from peripheral blood samples.Written informed consent was obtained from the parents or legal representatives of the involved patients.

| Literature review
We performed a systematic review of the literature on ADSL deficiency cases.We searched different online repositories (PubMed, EMBASE, and Google Scholar) for all the relevant articles.The search terms included "ADSL", "ADSL deficiency" and "Adenylosuccinate lyase deficiency".All the articles were screened by title and abstract by two reviewers (GC and SM).We then hand-searched relevant articles cited by the selected papers if they are not present in the initial search.All searches were carried out on October 10, 2022.We only included peer-reviewed case reports or series published in peer-reviewed journals in English, specifically reporting the genetic background and clinical features of the patients.The radiological data retrieved were reviewed by an expert child neuroradiologist.Patients without thorough clinical and/or genetic data were excluded from the synthesis (refer to Figure S1 for a flowchart of the screening process).

| Statistical analysis
Descriptive analysis was carried out using the median and interquartile range (IQR) for the quantitative variables and percentage values for the qualitative ones.The normality distribution for quantitative variables was assessed by the Shapiro-Wilk test.Pearson's chi-square test or Fisher's exact test was used to evaluate the association between categorical variables, while the non-parametric Kruskal-Wallis test was used to evaluate the differences between continuous variables and outcomes.After the Kruskal-Wallis test, for statistically significant results, the Dunn test was calculated for the comparison between the pairs of medians for the identification of significant differences.In addition, the survival analysis was performed by applying the Kaplan-Meier estimator and log-rank test for equality of survivor functions.Statistical significance was set at the level of ≤0.05.All analyses were performed using Stata software v17.1 (StataCorp, College Station, USA).

| Case series
Clinical data of the following patients are summarized in Table 1.As of the last examinations, both patients remained seizure-free under the administration of anti-seizure medications (refer to Figure 2 for a summary of EEG and MRI findings).As of the last neurological examination, the patient displayed limb and axial hypotonia, along with strabismus.Brain MRI at the age of 10 showed mild periventricular leukoencephalopathy and ventriculomegaly, with no overt cerebral or cerebellar atrophy.

|
Elevated urinary levels of SAICAr and S-Ado were observed in the patient.Whole-exome analysis confirmed the diagnosis, revealing the presence of the variants p.Y114H and p.R296W of the ADSL gene.These variants were inherited from healthy parents.The patient is currently 23 years old and still seizure-free with valproate monotherapy.

| Review of the literature
Clinical characteristics and demographics are summarized in Tables 2 and 3, with 88 individuals in the patient population, 81 retrieved from 29 articles in the literature, and 7 newly described patients (Figure S1).sometimes associated with regression of acquired skills, drug-resistant epilepsy, and often with spastic-dystonic tetraparesis.An exception is seen in five cases harboring the same homozygous variant (Pt.with developmental delay and autistic features within the first year and epilepsy onset in late childhood, achieving seizure freedom with appropriate treatment.The newly reported p.P24L variant's protein activity is not yet characterized.The four patients with p.P24L variants are compound heterozygous, and their phenotype, while consistent in terms of developmental delay and epilepsy presence, displays different degrees of severity even between siblings. 11Only a few patients were reported with additional variants, severely limiting the description of peculiar traits related to specific protein changes.

| Survival analysis
As expected, neonatal forms are associated with lower survival compared to the other two groups (Figure 3A).We computed the survival of the carriers of the four most frequent variants, that is, homozygous p.R426H, compound heterozygous p.Y114H, compound heterozygous p.R426H (excluding the p.Y114H and p.R426H carriers computed in the previous group), and homozygous p.D430N.The analysis revealed poor survival in the compound heterozygous p.Y114H and p.R426H groups and a better prognosis of the homozygous p.R426H and p.D430N carriers (Panel B, log-rank test = 17.89,P = 0.001).The neonatal onset, not accounting for mutation type, was associated with a poorer prognosis (Panel C).The presence of epilepsy is not associated with a statistically significant difference in terms of survival (Panel D).However, an earlier onset of epilepsy correlated with a worse prognosis (log-rank test = 8.92, P = 0.030).Seizures as the presenting condition and white matter abnormalities did not show significant differences in terms of survival (log-rank test = 0.37, P = 0.540; log-rank test = 1.07,P = 0.300, respectively).

| DISCUSSION
Although ADSL deficiency is classified into three broad phenotypes, the condition remains highly heterogeneous.The mechanisms by which ADSL deficiency can give rise to its symptomatology are not fully elucidated and may include deficiency of purine nucleotides, impairment of cellular bioenergetics, and toxic accumulation of SAICAr and S-ADO. 6Despite a diverse clinical presentation, epilepsy is present in a substantial portion of the affected patients (ie, 81.8%), with polymorphic and often intractable seizures, severely affecting the quality of life of the patients and    their caregivers.Earlier epilepsy onset and drug-resistant seizures appear to be more frequent in patients with type I and neonatal phenotypes and in patients harboring pathogenic variants encoding proteins with reduced residual activity.Different EEG patterns have been observed in ADSL patients: (i) a poor background organization with diffuse theta-delta activity was commonly reported; (ii) a burst suppression pattern or hypsarrhythmic pattern with epileptic spasms, especially at onset, has been described.As reported in our patients and by other authors, 12,13 the hypsarrhythmic pattern is generally responsive to ACTH or steroidal therapy, although the response is only limited in time.Also, burst suppression patterns have been reported.
5][16] Despite the wide clinical spectrum, the frontal or fronto-temporal predominance of EDs observed in all our cases and reported in previous literature, [14][15][16] could be considered a possible specific localization of EDs in this condition and seems to coincide with the areas that show the most significant atrophy on MRI. 17 Sporadically, occipital predominance or diffused spike-and-waves have also been observed, 15,18 (iv) sleep patterns were rarely reported, 19 and like our patients, the sleep structure was disorganized with no recognizable physiological sleep elements.No overt activation of spike-and-wave activity in sleep was previously reported; however, sleep recordings of our Pt. 3 and, to a lesser extent, Pts.1 and 2, showed EDs activation, also in bursts, evolving to a progressive impoverishment of the EEG background with loss of physiological sleep elements.EDs sleep activation in our Pt.3, despite not configuring a typical case of developmental and/or epileptic encephalopathy with spike-and-wave activation in sleep (DEE-SWAS) due to a different clinical evolution and genetic background, may account for his more severe cognitive phenotype in comparison with the sibling.Concerning the anti-seizure treatment for ADSL deficiency, to date, no standard of care can be recommended.However, in our milder patients (Pts.3-7), valproate, levetiracetam, and lamotrigine were found to be effective.Ketogenic diet, D-ribose, and S-adenosyl-Lmethionine 1,11,12,17,20 were implemented as possible therapeutic strategies in these patients with limited success (refer to Table S2).Clinical features, for example, psychomotor delay, intellectual disability, epilepsy, frequently associated with autistic traits, pyramidal and extrapyramidal signs, albeit common to many childhood encephalopathies, can be suggestive of this condition and configure a spectrum of disease presentation with differences in severity among the types of the disease.Also, imaging can aid in suspecting of ADSL deficiency, with similar features but with different grade of severity among the different phenotypes: cerebral, cerebellar atrophy, and white matter abnormalities tend to be more pronounced in Pts.1 and 2 (type I) compared to the patients we have described in families 2 and 3 (type II).These data were confirmed by the literature, where cerebral and cerebellar atrophy were reported in a significant proportion of patients, as well as white matter abnormalities (ie, periventricular or semioval center T2-hyperintensities).A previous review 17 specifically focusing on the MRI features of ADSL patients reported cerebral atrophy with frontal predominance as a common finding in older patients, in addition to cerebellar, specifically vermian, atrophy and white matter periventricular abnormalities.Such findings, in our cases and in literature, 17 tend to be more prominent in the most severe types of ADLS deficiency (type I) compared to the milder (type II) and more evident in older patients.They are not often reported in neonatal forms, in which cortical development abnormalities or cerebral hemorrhages are most commonly encountered, contributing to their poor prognosis and early death (preventing the establishment of overt cerebral atrophy).
Considering the features described in our new patients reported, in the literature revision, and the previous functional analysis conducted, the phenotype of the patients affected by ADSL deficiency seems to suggest a possible correlation with the residual activity of the ADSL protein. 7Patients harboring the p.Y114H variant, with minimal residual function, presented with neonatal forms of the disease, while patients with variants encoding for proteins with an almost normal residual function (eg, p.R190Q and p.D430N) presented with a relatively milder course.However, rarely, compound heterozygous patients can show a milder phenotype, for example, our Pt.7 (p.Y114H and p.R296W), suggesting the influence of the residual activity of the mutant protein encoded in the second allele in the establishment of the phenotype.Patients with the homozygous p.R426H variants, like our first family described, generally presented with a type I phenotype even if considerable variability could be observed between affected individuals, while patients with variants encoding for proteins with an almost normal residual function (eg, p.R190Q and p.D430N, like our reported patients) presented with a relatively milder course.Functional studies on new variants and the identification of new patients would be required to delineate a stronger phenotype-genotype correlation.

| Limitations
In our review, we included only a part of the literature published on ADSL patients because we focused only on peer-reviewed English-language journals and we included only studies or clinical cases reporting both genetic and clinical data.To date, many variants are present only in small groups of patients and are not fully clinically characterized.For a database of the variants identified up to June 2013, please refer to http:// www1.lf1.cuni.cz/ udmp/ ADSL/ .The low number of people with the disease and the high variability between the reported variants did not allow for a cluster analysis of the symptoms based on specific protein alterations.This analysis will be the goal of future work expanding the sample.

| CLINICAL RELEVANCE AND FUTURE DIRECTIONS
We present a series of previously unreported patients with ADSL deficiency with long-term follow-up documenting the electroclinical features of the syndrome and comparing our patients with previously published cases.Despite the fact that a precise genotype-phenotype association is not feasible due to the limited number of patients reported, clinical phenotype severity seems to correlate with residual protein activity.Also, clinical, neuroradiological, and neurophysiological data seem to display common features and developmental trajectories in ADSL patients: the development of cerebral and cerebellar atrophy and white matter periventricular abnormalities associated with an EEG pattern of EDs with frontal temporal predominance.In patients with psychomotor delay, epilepsy, prominent autistic features, and pyramidal-extrapyramidal signs, ADSL deficiency might be considered in the differential diagnosis of epileptic encephalopathies.

F I G U R E 1
Electroencephalogram (EEG) and magnetic resonance imaging (MRI) evolution of Pt. 2. Panel I-VI EEG of Pt. 2 from 12 months to 30 years of age (I) sleep EEG registration of Pt.2 (12 months year) shows hypsarrhythmia before the treatment with adrenocorticotropic hormone (ACTH); (II) awake EEG registration of Pt.2 after ACTH therapy shows a bilateral and symmetric 5-6 Hz background activity without epileptic discharges.(III) Awake EEG registration (13 years) showing high voltage, 1.5-2 Hz, spikes-and-waves predominantly over the frontal temporal regions.(IV) Awake EEG recording showing a disorganized background with spikes-and-waves predominantly on the frontal temporal regions and the start of a generalized seizure with clinical manifestations of staring spell and generalized stiffening.(V-VI) Awake EEG (24 and 29 years) showing high voltage, bilateral spikes-and-spikes, and slow waves on frontal temporal regions only mildly reduced in frequency and amplitude over sequential controls.Pt. 2's MRI at 10 (A and B) and 19 years of age (C and D).(A) (inversion recovery, coronal plane) and B (T1, sagittal plane) show diffuse and generalized brain atrophy and ventriculomegaly with a thin and mildly dysmorphic corpus callosum.Cerebellar and vermian atrophy are also evident.Anterior commissure, optic nerves and olfactory bulbs were intact.(C and D) (T2, transversal plane) show a progression of brain atrophy, mainly in the frontal and temporal lobes (coherent with epileptic discharge localization), with areas of periventricular T2 hyperintensity.An analogous progression of EEG and MRI findings was documented for Pt.1.

F I G U R E 2
Electroencephalogram (EEG) and magnetic resonance imaging (MRI) of Pts. 3 and 4. Panels I and II show the EEG of Pt. 3 and Pt. 4, respectively (both at 16 years): they display a common pattern of poor general organization, prevalence of diffuse theta activity, poor reactivity to eye closure, and rare spikes or spikes-and-waves with frontal predominance; poor sleep organization without recognizable sleep phases was also observed.Panel III (Pt.3, 12 years) shows an EEG during sleep with sequences of spike-and-wave activation in sleep evolving as the patient grew.Panel IV shows a sleep EEG of Pt.3 at 17 years in a dedifferentiation between awake and asleep states, with no recognizable sleep figures.Pt. 3 Brain MRI (16 years).(A) T1 sequence, sagittal plane: we can observe a thin corpus callosum and brainstem.Cerebellar atrophy is also noticeable.(B) T2 sequence, coronal plane: cerebral atrophy with enlarged ventricles.(C and D) T2 sequence, coronal and horizontal plane: marked cerebral and cerebellar atrophy are evident.

T A B L E 3
Clinical characteristics and symptom prevalence according to disease type (ie, I, II, or N).

T A B L E 3
(Continued) F I G U R E 3 Kaplan-Meier curves estimating survivals in different groups of patients.(A) Compares the different forms of the disease, showing lower survival rates in type N and I compared to type II patients.(B) Compares the most frequent variants observed in the sample, highlighting how lower survival is observed in p.Y114H heterozygous and p.R426H homozygous carriers.(C) Compares survival based on the age of onset, displaying how an earlier onset, not accounting for mutation type, was also associated with a poorer prognosis.(D) Compares the population of patients with epilepsy and the population without showing any statistically significant difference in terms of survival between the two population; however, we can notice a trend toward lower survival for patients with epilepsy.

3.3 | Family 2 (p.D430N, homozygous)
EEG and MRI findings).Whole-exome sequencing analysis revealed that both patients carried a homozygous variant c.1288G>A (p.D430N) in the ADSL gene, which they inherited from their healthy parents.Interestingly, only Pt. 4 displayed elevated urinary SAICAr and S-Ado levels.
Pt.5 and Pt.6 are French of Armenian descent.They presented with a psychomotor delay around the first year of life, more severe in Pt.5.Similar to previous cases, Pt. 6, the female patient, displayed a relatively milder phenotype compared to her brother.Pt. 6 did not exhibit overt autistic features, while Pt. 5 showed mild autistic traits.Neurological examinations were unremarkable except for the intellectual disability in both patients.Both developed epilepsy, respectively, at 11 and 9 years of age.
4.4| Family 3 (p.D430N, homozygous) The EEG showed diffused EDs more prominent in the frontal regions.Both patients achieved seizure freedom with valproate, associated with lamotrigine in Pt.6.Pt.6's EEG normalized after ASDs treatment. MRIshowed generalized atrophy in both patients, with white matter periventricular T2 hyperintensities in Pt. 5.Only Pt. 5 showed elevated urinary SAICAr and S-Ado.Whole genome analysis revealed the mutation c.1288G>A (p.D430N) in the ADSL gene, inherited from healthy parents.Currently, both patients are still seizure-free under anti-seizure drugs.3.5 | Family 4 (p.Y114H, p.R296W; compound heterozygosity) Pt. 7, born to non-consanguineous Italian parents, presented a relatively milder course of the disease.At around 18 months of age, the patient showed moderate psychomotor delay. Lter, autistic behavior with motor stereotypies and limited social interaction became evident.Seizures developed at approximately 10 years of age, with both generalized and focal seizures observed.The EEG displayed a mild alteration of the general organization with frontal temporal spikes.The patient achieved seizure freedom with valproate monotherapy.
Summary of the clinical features of reported cases.
T A B L E 2