PURA-Related Developmental and Epileptic Encephalopathy

Discussion

With this comprehensive study of 142 patients, we provide an outline of the phenotypic and genotypic spectrum of the PURA syndrome and we significantly expand the current patient population, suggesting that pathogenic variants in PURA might underlie ID and developmental and epileptic encephalopathy (DEE), more often than previously thought.

This study was done retrospectively and through clinician assessment of patients. When original EEG tracings were not available for review, EEG evaluation was based on EEG reports, discussing them, when necessary, with the referring physician. Unfortunately, longitudinal EEG data were not available; therefore, it has not been possible to evaluate the EEG evolution during the course of the disease.

Some data points are missing in some patients, although follow-up with clinicians was attempted. Patients were also assessed only once for inclusion; thus, longitudinal clinical data were not available in this study.

Most commonly, patients with PURA syndrome present with neonatal hypotonia, complicated by feeding difficulties, often requiring tube feeding, and respiratory distress.

The respiratory distress can be as severe as to cause respiratory insufficiency, requiring frequent recurrent pulmonary aspiration and often demanding mechanical ventilation because of the increased risk of central apnea, similar to the picture reported in patients with 5q31 microdeletions encompassing the PURA gene, who also feature a marked hypotonia at birth.⁸

Hypersomnolence and hypothermia further demonstrate the severity of the PURA syndrome. There were no significant differences between patients with and without neonatal hypotonia at inclusion, neither in the phenotypic appearances nor in the genetics. The hypotonia persists through childhood and adulthood, although in some patients, it gradually improves and motor skills continue to develop, eventually resulting in the acquisition of autonomous walking. This may reflect the steady increase of Pur-α during development, as seen in the mice.^3,19 The median age at inclusion was 6 years; thus, the estimated numbers are to be considered minimum values. In patients able to ambulate, gait is often ataxic; this finding can correlate with the fact that PURA expression peaks in the postnatal period, when interneuronal connections are being established, especially in the cerebellum.³ Gait disturbances were also found in the heterozygous mouse.⁴

Nonepileptic paroxysmal motor manifestations and pathologic startles were reported in a significant proportion of patients, both with and without epilepsy. In recent years, the occurrence of nonepileptic movement disorders in participants with DEE has been increasingly recognized.^20,21 Indeed, hyperkinetic movement manifestations, such as dystonia and choreoathetosis, are now considered a common phenotypic feature in individuals with FOXG1, GNAO1, SCN8A, CACNA1B, and STXBP1-related epilepsy-dyskinesia syndromes.^22,-,26 Hand stereotypies are commonly observed in children with autism or developmental delay, irrespective of precise etiology,²⁷ and “per se” they do not confer any specificity to affected patients; however, the definition of detailed accompanying phenotypes (such as hyperkinetic movements or hand stereotypies) can be important to characterize the disease spectrum and the early recognition of the PURA-related DEE, ultimately prompting referral of the patients for genetic testing.

A pathologic startle was observed in approximately 18% of our patients. At variance with our patients, the Pur-α_+/− mouse shows a trend toward a reduced startle response to acoustic stimuli, a finding considered unexpected by the authors of the study⁴ that requires to be further investigated, while the mouse also displayed occasional seizures on handling.³ Exaggerated startle attacks, described as sudden tonic postures with raising of bent arms over the head, flexion of the neck and trunk, in response to touch or sound stimuli, not associated with epileptic changes in the EEG,²⁸ have been reported in 38% of a cohort of children presenting with an encephalopathy associated with KCNQ2 pathogenic variants.²⁹ Our findings of a pathologic startle, previously unreported, might add to the constellation of clinical features that could contribute to an early diagnosis of PURA-related DEE.

In older patients, scoliosis is common because of truncal hypotonia. Other features include impaired intestinal motility (reflux and constipation most commonly), visual disturbances, and in a few patients hearing deficits as well. The underlying mechanisms for these features are unknown.

In our study cohort, 3 patients died because of respiratory failure and 1 because of SUDEP.^5,7,30 PURA knockout (KO) mice do not survive beyond 28 postnatal days³; however, an increased death rate is not observed in the heterozygous mice (PURA_+/−).³ Thus, we can speculate that if the human phenotype is comparable with the heterozygous mouse, the mortality seen in our cohort might be an expression of the increased mortality associated with severe developmental disorders and epilepsy in general and not linked directly to PURA. On the contrary, if the mortality rate observed here is indeed related to PURA-related DEE, it is lower than that reported in Dravet syndrome (15.84/1,000 person-years),³¹ whereas it is comparable with the mortality seen in other DEEs, such as SCN8A-related DEE (mortality 5.3%), which also shows similar causes of death.³² Lack of longitudinal follow-up in this study is a limitation in the assessment of the mortality. Further studies, in particular investigating the natural history of the PURA-related DEE, might be helpful to solve this issue and to establish the mortality rate of this DEE.

MRIs were normal in half of the patients, whereas the other half showed demyelination or loss of brain volume. In 1 patient (#54), follow-up MRI showed a normalization of myelination over time; therefore, we could speculate that myelination in patients with PURA syndrome is not absent but delayed. Further follow-up MRI data in additional patients will clarify this issue. Experimental studies in KO mouse have provided conflicting results, showing in some reports decreased signals from myelin and white matter tracts both in the subcortical white matter and in the cortex compared with wild-type littermates and reduced neuron density both in the cortex and in the cerebellum.³ Other studies in KO mice showed an enlarged brain size associated with a phenotype showing ataxia and continuous tremor.³³ The finding of a megalocephalic brain has been interpreted as resulting from a prolongation of neuronal precursor cell proliferation during postnatal development, an explanation that further supports the role of Purα in postnatal brain development.³

In a computational analysis of photographs of 34 PURA syndrome individuals, it was found that affected patients shared a similar gestalt, including a hypotonic face and high anterior hairline.⁷ In addition to those features, we expand the facial gestalt to include a flat nasal bridge with a widened and triangular nasal tip, thickened nostrils, a well-defined philtrum, heavy eyebrows, and periorbital fullness, present in a subset of individuals. Many syndromes have recognizable facial features that are highly informative to clinical geneticists.^34,-,36 Although PURA is not an easily recognizable syndrome, a detailed description of dysmorphic features and an understanding of how the features change over time can help clinicians identify further cases.

In total, 59.2% of the patients analyzed in this study have epilepsy, with age at onset ranging from the first day of life to 18 years. In the heterozygous mouse model, seizures/epilepsy are constant features in all animals.³ Most of the patients presented with a combination of seizure types that could be both focal and generalized in the same patient. Our results show that the most common seizure types are myoclonic, GTC, and focal with secondary generalization. A not negligible group of patients presented with reflex seizures whose semiology includes myoclonic, atonic, and “absence-like” manifestations. Various stimulus types (including visual, auditory, sensory, and painful stimulations), were reported as effective in triggering seizures. In some patients, a reflex mechanisms was observed only for a transitory period; therefore, we cannot exclude that the occurrence of reflex seizures in patients with PURA syndrome might be underestimated, particularly in patients with severe ID, high seizure frequency, co-occurrence of nonepileptic paroxysmal motor disorders, and limited monitoring.

The EEG also showed a mixture of focal/multifocal and generalized epileptic abnormalities. Of interest, an excessive activation of EEG epileptic discharges during sleep, resembling an ESES EEG pattern, was reported in 4 children; whether this sleep activation was associated with further cognitive/behavioral deterioration as in ESES syndrome was not possible to demonstrate because of the severity of the baseline cognitive status. Our findings suggest that PURA might add to the growing list of DEE-associated genes that can present during the course of the disease with an ESES-like EEG pattern.^37,38 Hypsarrhythmia and burst suppression were rarely observed. A syndromic classification that considered both the clinical and EEG features was possible only in 6 patients, with 4 of them presenting a clinical picture compatible with a Lennox-Gastaut syndrome and the other 2 with West syndrome. The remaining patients with epilepsy could be broadly diagnosed as presenting with a DEE, in most cases with a severe course.

In total, 40.9% of patients in our cohort did not present with epilepsy. However, because of the broad age range of epilepsy onset (from the first day of life up to 18 years) and the age at inclusion in this study, only 1 patient without epilepsy was older than 18 years. Thus, we cannot exclude that a proportion of nonepileptic patients will develop epilepsy later in the course of their disease, in particular those nonepileptic patients who already present epileptic abnormalities in their EEG. This hypothesis may be supported by the statistical significant difference in the age at inclusion in our study of the subgroup of patients with epilepsy who were older than the subgroup without epilepsy. The nonepileptic patient older than 18 years was retrieved in the literature, and he was a 19-year-old man who presented with developmental delay since birth; he was able to walk by the age of 4 years and to communicate with a one-two word vocabulary.¹³ He had a normal MRI and normal EEG (the last one at 14 years of age).¹³ He carried a de novo recurrent missense variant, p.(Ile188Thr), as well as an Xp22.31 duplication.¹³ In the literature, another patient with the same missense variant also had no seizure activity at the age of 8 years,² whereas a third patient with a frameshift variant at the same position had GTCs with an unknown age at onset.⁵ Thus, at present, an estimation of the prevalence of epilepsy in the PURA population is not reliable because most of the reported patients are children, who could still present with epilepsy at a later age. In line with this, pediatric epilepsy cohort studies have not identified PURA as one of the more common causes of epilepsy in infancy.³⁹ However, in a recent study from our center, we identified a pathogenic variant in PURA as the underlying cause of epilepsy in 1% of adult patients.⁴⁰

Our results show that 66.6% of the patients with PURA syndrome suffer from drug-refractory epilepsy. Although some ASMs, such as VPA, LEV, and TPM, showed favorable results in some patients, the same drugs were ineffective in other patients, currently making treatment recommendations for patients with PURA syndrome difficult. Further studies elucidating the underlying pathophysiologic mechanisms in PURA-related epilepsy will be crucial, including (heterozygous) animal models to test possible novel drugs and to identify possible precision medicine approaches.

In this study, we expand the genetic landscape of PURA by including 38 new variants. In all patients where segregation analysis was completed, the variants arose de novo. In addition, most of the patients have variants that are loss of function per se, such as frameshift or indel variants. This is supported by a pLoF (predicted loss-of-function) score of 8.9 (pLi 0.94), suggesting that the PURA gene is intolerant to loss of function variants (gnomad.broadinstitute.org/). Thus, haploinsufficiency seems to be the most likely functional defect underlying the disease. Although missense variants have not been tested functionally, the evidence that patients with missense variants display phenotypes similar to the patients with loss of function variants points toward similar functional effects.

Most of the variants were located within the PUR repeats, which underline the importance of these repeats for the function of PURA.⁴¹ Some variants, both nonsense and missense, are still located outside the PUR repeats, suggesting that these areas of the gene are also crucial for the correct functioning of PURA.

Phenotypic variability was seen even within variants; where the same variant was seen in patients with and without epilepsy. This might reflect other factors, such as epigenetic modulators, or it might be simply due to a difference in age of the patients (see above).

Previously, a single patient with a variant in the C-terminal of the protein was described with a milder phenotype compared with other patients, which led the authors to suggest that it is unlikely for variants in the far end of the C-terminal to affect protein folding or nucleic acid binding.⁷ However, our results do not support this hypothesis because also patients with variants in this part of the gene (such as #42) seem to display an equally severe PURA phenotype. There were also no apparent differences between variants in the 5′ and 3′ end of the protein. No clear genotype-phenotype associations emerged from the analysis of our cohort.

Our study further defines and expands the phenotypic and genotypic spectrum of the PURA-related DEE. Some clinical features, especially in the neonatal age, including hypotonia, feeding, and respiratory difficulties, are characteristic and should raise the suspicion of PURA, prompting genetic screening. In our cohort, more than 60% of patients have epilepsy, characterized by a very wide range of age at onset, from the neonatal period up to early adulthood; therefore, it cannot be excluded that a further fraction of patients, still in their childhood and not presenting epilepsy yet, will develop epilepsy later in their disease course. The epilepsy featured a wide spectrum of seizure types, including both focal, generalized seizures, and reflex seizures, refractory to currently available ASMs in the great majority of patients. Finally, our study failed to identify genotype-phenotype associations. Additional studies investigating the functions of PURA and the pathophysiologic mechanism underlying the PURA-related DEE are needed to guide the search for more effective and possibly targeted treatments.