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Abstract

  1. Top of page
  2. Abstract
  3. What this paper adds
  4. Method
  5. Molecular analyses
  6. Discussion
  7. Acknowledgements
  8. References

Aim  To describe a spectrum of intracerebral large artery disease in Aicardi–Goutières syndrome (AGS) associated with mutations in the AGS5 gene SAMHD1.

Method  We used clinical and radiological description and molecular analysis.

Results  Five individuals (three males, two females) were identified as having biallelic mutations in SAMHD1 and a cerebral arteriopathy in association with peripheral vessel involvement resulting in chilblains and ischaemic ulceration. The cerebral vasculopathy was primarily occlusive in three patients (with terminal carotid occlusion and basal collaterals reminiscent of moyamoya syndrome) and aneurysmal in two. Three of the five patients experienced intracerebral haemorrhage, which was fatal in two individuals. Post-mortem examination of one patient suggested that the arteriopathy was inflammatory in origin.

Interpretation  Mutations in SAMHD1 are associated with a cerebral vasculopathy which is likely to have an inflammatory aetiology. A similar disease has not been observed in patients with mutations in AGS1 to AGS4, suggesting a particular role for SAMHD1 in vascular homeostasis. Our report raises important questions about the management of patients with mutations in SAMHD1.


Abbreviation
AGS

Aicardi–Goutières syndrome

What this paper adds

  1. Top of page
  2. Abstract
  3. What this paper adds
  4. Method
  5. Molecular analyses
  6. Discussion
  7. Acknowledgements
  8. References
  •  Mutations in SAMHD1 can be associated with intracerebral large artery disease.
  •  SAMHD1 is implicated in vascular homeostasis.

Aicardi–Goutières syndrome (AGS) is a genetically determined encephalopathy, which, in its classical presentation, shows phenotypic overlap with the sequelae of congenital infection.1 It is a genetically heterogeneous disorder caused by mutations in any of the genes encoding the 3′ to 5′ exonuclease TREX1 (AGS1),2 the three non-allelic components of the RNASEH2 endonuclease complex (AGS2, 3, and 4),3 and the uncharacterized SAMHD1 protein (AGS5).4 With identification of the genetic basis of AGS, the clinical phenotype has expanded to include a milder disease with later onset in many cases.5,6 Additionally, mutation screening of AGS-related genes has shown an association with a wider spectrum of apparently distinct neurological and non-neurological phenotypes, including retinal vasculopathy with cerebral leukodystrophy,7 familial chilblain lupus,8 systemic lupus erythematosus,9 and non-specific inflammatory arthropathy.10

Neuropathological data,11 the sometimes patchy white-matter changes seen on brain imaging, and the observation of a vasculitis12 on biopsy of the chilblain lesions experienced by 40% of patients, suggest that an inflammatory disturbance of vascular homeostasis may be central to the pathogenesis of AGS. Here we report five patients with a clinical diagnosis of AGS who developed intracranial large artery disease that was primarily occlusive in three patients, and associated with aneurysm formation in two further patients. All five affected individuals were shown to harbour biallelic mutations in SAMHD1.

Method

  1. Top of page
  2. Abstract
  3. What this paper adds
  4. Method
  5. Molecular analyses
  6. Discussion
  7. Acknowledgements
  8. References

Appropriate written informed consent was obtained from the parents of the patients for inclusion in this study, which was approved by the Leeds (East) Multi-centre Research Ethics Committee. Clinical, radiological, and mutation data are summarized in Table I.

Table I.   Clinical, radiological, and genetic data of the five patients with Aicardi–Goutières syndrome demonstrating intracerebral large vessel disease
Patient12345
SexFMFMM
EthnicityWhite BritishAfricanWhite CanadianWhite ItalianAshkenazi Jewish
Parental consanguinityNoNoNoNoNo
Age at chilblain onset2y6y2y13y20mo
SeveritySevere (monthly prostacycline infusions)Severe (autoamputation of right ring finger)SevereSevereSevere (autoamputation of toe and finger tips)
BiopsyNoNoLeukocytoclastic vasculitisLeukocytoclastic vasculitisNo
GlaucomaNoYesNoNoNo
SAMHD1 mutation(s)Homozygous: c.602T>A (I201N)Homozygous: IVS13+1G>TCompound heterozygous: c.427C>T (R143C) and c.602T>A (I201N)Homozygous deletion of exons 12–16Compound heterozygous: c.649insG (F217CX) and deletion exon 1
Degree of intellectual disability Intracerebral vessel diseaseModerate. Good language, vision, and hearingModerate. Speaking sentencesSevereSevereSevere. Some communication abilities
StenosisYesYes, with evidence of infarctsNoNoYes
CollateralsYes NoNoYes
Aneurysmal formationNo YesYesNo
StatusDied age 7y of intraventricular haemorrhageAlive at age 10yDied age 9y of intracerebral haemorrhageIntracerebral haemorrhage at age 13y; alive at age 14yAlive age 25y

Patient 1

This female was born at term by vaginal delivery. There was no parental consanguinity. By 9 months of age she was demonstrating a motor delay and examination revealed spasticity in four limbs. Computed tomography (CT) of the brain showed bilateral basal ganglia calcification (Fig. 1a). There was no evidence of intrauterine infection. Her head size was normal and remained so subsequently. However, other growth parameters fell below the 3rd centile from an early age: at age 5 years 6 months her weight was 13kg (0.4th centile) and her length was 94.7cm (5cm <0.4th centile).

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Figure 1.  Computed tomography of patients (a) 1, (b) 3, and (c) 4 at 8 years, 7 years, and 1 month respectively, showing symmetrical basal ganglia calcification, and low attenuation of the frontal white matter in (b) and (c). Calcification at the white–grey matter junction is also seen in c. (d) T2-weighted magnetic resonance image (MRI) of patient 1 at 8 years showing extensive basal collaterals and an old infarct in the left occipital lobe. (e) T2-weighted MRI of patient 2 at 7 years showing mature infarcts in the right middle and right and left anterior cerebral artery territories. (f) T2-weighted MRIs of patient 3 at 7 years and (g) patient 4 at 13 years showing subcortical and cortical atrophy, with diffuse high signal in frontal white matter.

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From childhood she had chilblains and Raynaud-type phenomenon, showing cyanosed tips of the fingers and toes on exposure to cold with occasional skin breakdown (Fig. 2a). She was referred to the rheumatology service at age 5 years because of this problem. The severity of her peripheral ischaemic changes led to treatment with monthly prostacycline infusions.

image

Figure 2.  (a) Hands of patient 1, and (b) feet of patient 2 and (c) patient 3 showing significant chilblain lesions. Note the loss of tissue in patient 2.

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At the age of 6 years she had good language, vision, and hearing, although she attended a special school and was functioning in the moderate learning difficulties range. Her mobility was limited by quadriparesis, necessitating the use of a wheelchair.

Because of the chilblains and intracranial calcification, magnetic resonance imaging (MRI) of the brain was performed at age 7 years. This revealed bilateral severe stenosis of the supra-clinoid internal carotid and the middle and posterior cerebral arteries, with profuse collaterals around the basal ganglia and thalami, and lacunar infarcts in the right centrum semiovale and left posterior parietal and occipital lobes (Figs 1d and 4a). These findings were interpreted as consistent with an occlusive vasculopathy and the secondary development of moyamoya collaterals.

image

Figure 4.  (a) Magnetic resonance angiogram (MRA) of patient 1 (lateral view) showing stenoses of terminal internal carotid and basilar arteries with extensive collateral formation (moyamoya). (b) MRA of patient 2 (coronal view) showing narrowing of the right terminal and proximal anterior and middle cerebral arteries. Note the absence of collaterals in this patient. (c) Digital subtraction angiography in patient 4. Aneurysms, marked by crosses, are seen at the junction of the right middle cerebral artery with the anterior temporal artery, a distal branch of the right middle cerebral artery after its bifurcation, and the junction of the right pericallosal artery with the callosal marginal artery junction.

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She was started on aspirin and referred for consideration of surgical revascularization. However, she then had an intraventricular haemorrhage. Despite neurointensive care it was not possible to wean her from the ventilator. She died soon after.

Patient 2

This male was the product of the first pregnancy of non-consanguineous parents. The pregnancy was uneventful and he was delivered vaginally at term with a birthweight of 2600g (2nd centile).

He appeared well until age 4 months, when he developed multiple generalized convulsive seizures. Brain CT showed intracranial calcification. A serological screen for intrauterine infection was negative.

At 13 months of age his irides had a greyish tinge and he was identified as having raised intraocular pressure, which was treated initially with eye drops and subsequently by trabeculectomy.

He was diagnosed with cerebral palsy (CP) at 5 months of age. He sat independently at age 6 months, commando-crawled at 2 years, and began to pull himself to stand at 3 years of age. However, he never walked. He had some single words at age 3 years and was speaking simple sentences at 5 years of age. He was doubly incontinent. His visual acuity was difficult to assess but he had normal hearing.

At the age of 6 years he began to show ulceration of the tips of his fingers, toes, ears, and nose, which was exacerbated by cold weather (Fig. 2b).

On examination, his weight was 15.5kg (3.5kg below the 3rd centile) and his head circumference was 44.5cm (4.5cm below the 2nd centile). He had healed vascular lesions on his fingers and toes. His tone was generally increased, with brisk reflexes and equivocal plantars. He had contractures in his right upper limb at the elbow, and at both knees.

Brain MRI at age 5 years showed extensive right middle and anterior cerebral artery territory infarcts associated with volume loss of the right cerebral hemisphere, and left anterior cerebral artery territory infarct (Fig. 1e). There was evidence of periventricular signal change and cysts. The circle of Willis showed stenosis of the supra-clinoid internal carotid arteries bilaterally, with attenuation of the right middle cerebral artery branches (Fig. 4b).

Patient 3

This female was born to unrelated parents at 41 weeks’ gestation weighing 3235g (25th centile). Examination at birth was normal; she had a head circumference on the 50th centile.

By 4 months of age she was showing signs of developmental delay, microcephaly, and cortical visual impairment. CT brain imaging revealed periventricular calcification with ventriculomegaly. A diagnosis of CP secondary to intrauterine infection was considered. However, a toxoplasmosis, rubella, cytomegalovirus and herpes simplex (TORCH) screen was negative and there was no evidence of chorioretinal disease or optic atrophy.

At the age of 2 years she developed violaceous discoloration of the fingers and toes (Fig. 2c). This was aggravated by cold weather but never resolved completely. There was recurrent ulceration of the digital skin. She also demonstrated purpuric annular lesions on the anterior lower legs, hands, and feet, violaceous atrophic lesions on her chin and eyelids, and an erythematous papular eruption on the sides of the chest.

A skin biopsy from a lesion on the left lower leg showed a leukocytoclastic vasculitis. A skin biopsy from the chest showed extensive mucin deposition between collagen bundles in the dermis and a mild perivascular lymphocytic infiltrate.

Laboratory investigations found a persistently elevated erythrocyte sedimentation rate, a mild neutropaenia, elevated antinuclear antibody titre, negative anti-double-stranded DNA antibody and anti-extractable nuclear antigen antibodies, elevated anti-cardiolipin immunoglobulin M, negative von Willebrand antigen, weakly positive antineutrophil cytoplasmic antibody titre, and normal C3, C4, and CH50 complement levels. Cryoglobulins were not detected.

During the next 6 years there was episodic progression of her skin disease, with development of ischaemic ‘atrophie-blanche’ changes on the feet and hands. She had livedo reticularis on her chest and more extensive violaceous atrophic patches on the chin, nasolabial folds, and eyelids. She was treated intermittently with prednisone, hydroxychloroquine, azathioprine, aspirin, and warm gloves and footwear. She had two episodes of streptococcal cellulitis, which required hospitalization for intravenous antibiotic therapy.

At 6 years of age she was found to have progressive loss of neurological function, inconsistent with a diagnosis of CP. CT and MRI of the brain showed symmetrical bilateral basal ganglia calcification, calcification at the white–grey matter junction, and low attenuation in the frontal white matter with subsequent extensive loss of white matter (Fig. 1b,f). Examination of the cerebrospinal fluid (CSF) showed three white cells per cubic millimetre; CSF interferon alpha levels were not measured.

At 9 years of age she had a large subarachnoid haemorrhage. Neuroimaging revealed progressive ventriculomegaly, probably secondary to subarachnoid haemorrhage, and an aneurysm of the left internal carotid artery (Fig. 3a). She died after a further haemorrhage. Post-mortem examination showed a vasculitis of intracerebral and leptomeningeal vessels, and calcification in the walls of small arteries and veins. There was evidence of a ruptured aneurysm at the origin of the left middle and anterior cerebral arteries, and an unruptured aneurysm of the right middle cerebral artery.

image

Figure 3.  Computed tomography (CT) of (a) patient 3 aged 9 years showing extensive subarachnoid haemorrhage and aneurysm of the left terminal internal carotid artery. (b) CT of patient 4 aged 13 years demonstrating right intraparenchymal haematoma with intraventricular extension.

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Patient 4

This male infant, born at term after an uneventful pregnancy, presented at the age of 1 month with hypotonia, dystonic posturing, generalized seizures, gastro-esophageal reflux, and a refusal to feed. CT of his brain revealed bilateral punctuate calcifications in the basal ganglia and periventricular white matter, ventricular enlargement, and periventricular frontal and temporal white-matter hypodensities (Fig. 1c). Brain MRI at age 6 months showed a periventricular leukoencephalopathy involving the frontal and temporal lobes more than the parietal and occipital lobes. Serology for TORCH infections was negative and a metabolic work-up, including muscle and skin biopsies for mitochondrial studies, was unremarkable. The CSF was normal, with no lymphocytosis.

At the age of 13 years the patient presented with painful bluish-red swelling of the fingers, toes, and ear helices. The lesions were exacerbated by cold. Skin biopsy from affected digital skin showed a leukocytoclastic vasculitis.

Neurological examination revealed microcephaly, strabismus, spastic tetraparesis, hypertonia of the limbs, truncal hypotonia, and dystonic movements. Repeat brain MRI showed a degree of atrophy with confluent areas of high T2 signal throughout the hemispheric white matter (Fig. 1g). The corpus callosum, internal capsule, and cerebellum were spared.

Three months later the child presented with a subacute onset of coma. Ten days after his deterioration began, CT and MRI showed a deep right temporoparietal intracerebral haematoma with intraventricular extension (Fig. 3b). Angiography revealed three aneurysms arising from the junction of the right middle cerebral artery with the anterior temporal artery, from a distal branch of the right middle cerebral artery after its bifurcation, and at the junction of the right pericallosal artery and callosomarginal artery (Fig. 4c).

Patient 5

This male was born to non-consanguineous parents of Ashkenazi–Jewish ancestry. He was delivered at term with normal growth parameters. During early infancy he was thought to show possibly delayed development. At age 7 months he developed an acute left hemiparesis. At 11 months of age he experienced another episode of left-sided weakness. A thrombosis of the right internal carotid artery was diagnosed on clinical and radiological grounds. His condition was stable until age 25 years, at which time he was tetraplegic, could not speak, read, or write but did hear well, would laugh appropriately at certain jokes and could communicate using a talking-board. He had a head circumference less than three standard deviations below the mean. He was fed by gastrostomy, which had been placed at age 18 years because of poor weight gain.

From the age of 20 months he demonstrated Raynaud phenomenon of the feet, hands, and ears, which was much worse in the winter months. There was frequent skin breakdown and eventual tissue loss from the distal ends of the finger and toe tips. He also showed more widespread skin involvement described as cutis marmorata.

Brain imaging was undertaken at the age of 25 years because of an unexplained deterioration in skills. No films are available for this patient. However, brain MRI was reported to show significant atrophy, with evidence of old infarcts in the frontoparietal and occipital lobes. Additionally, lacunar infarcts were noted in the basal ganglia. Magnetic resonance angiography indicated severe narrowing and obstruction of the upper siphon of the internal carotid arteries bilaterally, with bilateral filling through collateral arteries of the middle cerebral and anterior cerebral arteries. There was significant narrowing and paucity of middle cerebral artery branches on the left side compared with the right, whereas the upper part of the right vertebral artery seemed obstructed, with obstruction also of the basilar artery. The posterior cerebral arteries appeared attenuated. The imaging was considered consistent with moyamoya syndrome.

A male sibling of this patient was delivered after an uneventful pregnancy. At age 3 months glaucoma was diagnosed, at which time he was already showing severe development delay. Brain CT at 18 months of age revealed calcification of the basal ganglia. He developed Raynaud disease of the hands in infancy. He died at age 26 years of pneumonia. No further information is available.

Molecular analyses

  1. Top of page
  2. Abstract
  3. What this paper adds
  4. Method
  5. Molecular analyses
  6. Discussion
  7. Acknowledgements
  8. References

Genomic DNA was extracted from peripheral blood leukocytes using standard techniques. Primers were designed to amplify the coding exons of SAMHD1 as described previously.4 Purified polymerase chain reaction (PCR) amplification products were sequenced using BigDye terminator chemistry (Life Technologies, Carlsbad, CA, USA) and an ABI 3130 DNA sequencer. Mutation description is based on the reference cDNA sequence NM_015474, numbering relative to 1=A of the initiator codon. The following primers were used for detection of the exon 1 deletion: (forward) 5′-ttcccttttctgcaatgctt-3′ and (reverse): 5′-ccggccaagagaaactttatt-3′, giving a PCR product of 501 base pairs.

Molecular genetic results

Patient 1 was homozygous for the transversion c.602 T>A in exon 5 of SAMHD1, resulting in a substitution of an asparagine for an isoleucine at amino acid 201 (I201N). Both parents were heterozygous for this change. The same mutation was seen in patient 3. The isoleucine residue at this position is conserved to the starfish Nematostella vectensis.

Patient 2 was homozygous for a splice-site mutation at the conserved +1 position in intron 13 to 14 (IVS13+1G>T), which is predicted to result in a splicing defect. Both parents were heterozygous for the same mutation.

Patient 3, as previously reported,4 was found to be compound heterozygous for the C>T transition at c.427, resulting in a substitution of an arginine for a cysteine at amino acid 143 in exon 4 (R143C), and the same T>A transversion at c.602 (I201N) as seen in patient 1. As with the I201 residue, the arginine at position 143 is conserved from humans to starfish. Father and mother carried the R143C and I201N mutations respectively.

Patient 4 demonstrated non-amplification of exons 12 to 16 of SAMHD1, despite good amplification of other exons using DNA from this patient, and amplification of both parents and control DNA for all exons of SAMHD1. Attempts to use PCR across the presumed deletion breakpoint were unsuccessful.

Patient 5 was compound heterozygous for a single base-pair insertion of a G at c.649 in exon 6, resulting in an insertion of a cysteine and truncation of the protein at amino acid 217 (F217CX). Additionally, this patient was refractory for amplification of exon 1 of SAMHD1, despite good amplification of other exons using DNA from this patient, and amplification of both parents and control for this exon. Amplification by PCR across the breakpoint revealed an 8984 base-pair deletion starting upstream of the promoter and ending in intron 1. The mother and father carried the deletion and the insertion respectively.

Discussion

  1. Top of page
  2. Abstract
  3. What this paper adds
  4. Method
  5. Molecular analyses
  6. Discussion
  7. Acknowledgements
  8. References

Here we describe five patients, four with confirmed mutations and a fifth likely to harbour a homozyguous intragenic deletion in the AGS5 gene, SAMHD1, all of whom had intracerebral large artery disease in association with a cutaneous vasculopathy. Three patients showed multiple and extensive stenoses of intracerebral large arteries with, in two patients, evidence of basal collaterals consistent with a radiological diagnosis of moyamoya syndrome. Two further patients showed intracranial aneurysms without evidence of occlusive disease. One child, for whom imaging was not available, presented at 7 months of age with acute hemiparesis, reportedly due to thrombosis of the right internal carotid artery. Three patients experienced intracerebral haemorrhage before the age of 14 years, which was fatal in two.

We note a previous report of a patient demonstrating an occlusive vasculopathy with moyamoya collaterals and peripheral vascular occlusive disease, presenting at 13 years of age with acute hemiplegia.13 This male, resident in Australia, had a history of chilblains of the hands and feet from the age of 6 months which were more severe in cold weather, and loss of tissue from the tips of his ears. Carotid angiography revealed narrowing of the supra-clinoid portions of both internal carotid arteries, occlusion of the anterior and middle cerebral vessels just distal to their origins from the internal carotid arteries, and a vast network of fine collaterals in the region of the basal ganglia. Of note, arteriography of the left forearm and hand showed occlusion of the ulnar artery and very slow circulation in the hand, fingers, and thumb. We speculate that this patient’s disease was due to mutations in SAMHD1.

Two of our patients were considered to have moyamoya syndrome on radiological grounds, with occlusive disease of the large vessels of the circle of Willis, and the compensatory development of a collateral circulation. Although most cases of moyamoya disease have no definable explanation,14 rare monogenic associations are recognized, including sickle cell disease, neurofibromatosis type 1, homocystinuria, microcephalic osteodysplastic dwarfism, ACTA2-related disease, and Schimke immunosseous dysplasia. Our findings suggest that disease due to mutations in SAMHD1 should now be added to this list. The spectrum of associated conditions reinforces the importance of appreciating that moyamoya remains a radiological label, and is likely to represent the end result of more than one disease process.

All five of the patients we describe experienced significant chilblain-like lesions, a feature reported in association with mutations in all five known AGS-associated genes. Two patients demonstrated a leukocytoclastic vasculitis on biopsy, characterized by the presence of fibrinoid necrosis, red-cell extravasation, and neutrophil infiltrate. This well-defined histopathological designation is considered indicative of a true vasculitis. Taken together with the reported findings of an intracranial vasculitis on post mortem of patient 3, it would seem likely that the cerebral arteriopathy we describe has an inflammatory basis. Akin to ACTA2-related vascular disease,15 a non-inflammatory vasculopathy, the skin of patients with SAMHD1 mutations may reflect a more diffuse involvement of multiple arterial beds, predisposing to both occlusions and aneurysm formation.

We previously described glaucoma in three children with AGS.16 In this report, one patient and a sibling of another (patient 5), showed congenital glaucoma, possibly caused by an acquired abnormality of the microvasculature of the trabecular network at the angle of the anterior chamber.

Previous, limited, neuropathological data have suggested that AGS may represent a primary microangiopathy, a contention supported by the pattern of changes seen on brain imaging, and the frequently reported finding of a vasculitis on biopsy of the chilblain lesions experienced by 40% of patients with AGS. These observations suggest that an inflammatory disturbance of vascular homeostasis may be central to the pathogenesis of AGS. Interestingly, heterozygous mutations in the AGS1 gene TREX1 cause the adult-onset disorder retinal vasculopathy with cerebral leukodystrophy, previously known as hereditary endotheliopathy, retinopathy, and nephropathy.7 The neurological manifestations of retinal vasculopathy with cerebral leukodystrophy include transient ischaemic attacks and strokes, and the mass lesions observed on brain imaging demonstrate a coagulative necrosis secondary to an obliterative vasculopathy with minimal inflammatory infiltrate on biopsy. Additionally, the ophthalmological findings seen in retinal vasculopathy with cerebral leukodystrophy, which include capillary dropouts with prominent juxta-foveolar capillary obliteration and telangiectasias, are also consistent with a primary vasculopathy.17 Although the function of SAMHD1 remains unknown, evidence suggests that, like TREX1, it plays an important role in innate immunity and inflammation.18

Clinical and laboratory data have previously indicated that, after an initial subacute encephalopathic period, the disease process in AGS is most frequently non-progressive.5 However, in the patients described here there was definite evidence of ongoing pathology, and our report raises important questions about the management of patients with mutations in SAMHD1. In practical terms both the occlusive and aneurysmal arteriopathies described here might be amenable to treatment (revascularization for the former, and coiling or clipping for the latter). Moreover, the likely inflammatory basis of the arteriopathy suggests that immunosuppression may have a role in management. A key question is whether inflammatory disease is active at the time of clinical presentation, or whether the arterial abnormalities observed represent the end result of a now-quiescent inflammatory process. Given the potential for intervention, it could be argued that individuals with mutations in SAMHD1 should be actively screened for intracranial arteriopathy. Cognisance would need to be taken of the overall clinical presentation when making decisions about screening and intervention.

Interestingly, we have never observed large artery disease in association with mutations in AGS1-4, perhaps indicating a particular role for SAMHD1 in blood vessel integrity and homeostasis. More generally, following our recent description of progressive arthropathy with distal joint contractures and painful mouth ulcers in association with bialleic SAMHD1 mutations,10 this report suggests the need to consider mutation analysis of SAMHD1 in overlapping inflammatory phenotypes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. What this paper adds
  4. Method
  5. Molecular analyses
  6. Discussion
  7. Acknowledgements
  8. References

We are very grateful to the families for their involvement in our research and to the clinicians who provided us with clinical data and samples. YJC acknowledges the Manchester National Institute for Health Research Biomedical Research Centre.

References

  1. Top of page
  2. Abstract
  3. What this paper adds
  4. Method
  5. Molecular analyses
  6. Discussion
  7. Acknowledgements
  8. References