Abstract
INTRODUCTION
Mutations of the APP gene encoding the amyloid-β protein precursor are responsible for increased production of the amyloid-β (Aβ) peptide and/or the production of a more aggregation-prone form. This results in increased aggregation of Aβ oligomers in the brain parenchyma, a key event in Alzheimer’s disease (AD) pathogeny. AD pathology is frequently associated with Aβ-related cerebral amyloid angiopathy (CAA), which results from the deposition of the Aβ peptide in the walls of cortical and leptomeningeal vessels. CAA is mainly responsible for an increased risk of hemorrhagic strokes, including cortical and subcortical microbleeds, lobar hematomas, and subarachnoid hemorrhages. Duplications encompassing APP and several APP point mutations, located in the region coding for the Aβ peptide (i.e., Dutch, Italian, Arctic, Iowa, Flemish, and Piedmont), are primarily associated with severe CAA with an early onset. CAA can be the presenting diagnosis, before AD occurs or not (for review, see [1]). In two families, a very specific imaging phenotype was associated with an APP mutation known as Iowa [2]. In both pedigrees [3, 4], cortical occipital calcification was identified on computerized-tomography (CT) scans. Recently, two novel families from Ireland and Poland were identified with familial CAA related to this specific mutation [5, 6]. Cortical calcifications were only reported on CT scans of patients with an APP Iowa mutation in the context of familial CAA.
Our aim was to report the clinical, imaging, and biological features associated with APP mutations identified in France in the context of CAA. For that purpose, we reviewed cases sent to two genetics laboratories for APP mutation screening in this context. We provide a literature review of other patients carrying the same mutations as those identified in our patients and discuss the putative meanings of cortical calcifications associated with CAA.
MATERIALS AND METHODS
Inclusion criteria
We reviewed medical records of unrelated patients for which the corresponding physician (most frequently a Neurologist) requested the analysis of the APP gene in the context of CAA during the past 15 years. Blood samples were referred to one of the two French centers performing such sequencing in France: the National Reference Center for Young Alzheimer Patients (CNR-MAJ), Rouen, and the Department of Neurovascular Genetics, Lariboisière Hospital, Paris, France. We retained only patients with an age of onset before 66 years and fulfilling the other criteria of probable, probable with supporting pathology, or definite CAA [7]. Note that fulfilling Boston criteria requires an age of 55 or more. This criterion was not taken into account as this study focused on young patients. As patients with or without family history of dementia or stroke can carry an APP mutation in the presence of a censoring effect or a de novo occurrence of the mutation, we included patients regardless of their medical family history.
Patients with an APP duplication fulfilling the criteria of AD and CAA were not included in this study as they were already previously reported [8].
After the identification of APP mutations, we included relatives showing neurological symptoms for clinical and radiological description.
Cerebrospinal fluid (CSF) AD biomarkers are reported, when available. CSF samples were collected by lumbar puncture and stored in polypropylene tubes. Samples were analyzed with the INNOTEST® assays (Fujirebio, USA) according to the manufacturer protocols. Following thresholds were used for abnormal values: Aβ42<500 pg/mL; Tau>350 pg/mL, P-Tau>60 pg/mL.
Genetic analyses
All patients and controls gave informed, written consent for genetic analyses. DNA was extracted from whole blood. We sequenced exons 16 and 17 of the APP gene and performed APOE genotyping by Sanger sequencing. Mutation nomenclature refers to NM_000484.3 for APP. We also analyzed six microsatellite-containing regions surrounding the APP gene by PCR in the proband of the original Iowa pedigree [2] and the two patients from the French pedigree with the same Iowa, p.Asp694Asn, mutation. The following microsatellites were studied, from centromere to telomere (21q21.3 region): D21S265, D21S1268, D21S1253, D21S1443, D21S1896, and D21S1435.
Review of literature
We performed a systematic search on PubMed (accessed in May 2016) using the following keywords: APP mutation or cerebral amyloid angiopathy and Flemish or Iowa or Italian or D694N or E693K or A692G or D23N or E22K or A21G. We then manually curated all results and kept studies reporting at least one mutation carrier. Among the reports from the literature and our studied families, we retained all ascertained carriers of the mutations, obligate carriers, and first-degree relatives of mutation carriers with a phenotypic description reminiscent of CAA and/or AD only when sufficient clinical information was provided. Presymptomatic carriers were not included with the exception of the lately reported family from Spain where the genetic diagnosis was first made in a presymptomatic carrier [9].
RESULTS
Sixty-one unrelated patients fulfilled the criteria for probable (n = 56), probable with supporting pathology (n = 2), or definite (n = 3) CAA with an age of onset <66 years (36 males, 25 females). The mean age at which a first neurological sign was noticed was 55.56 years (±6.98, SD). Fourteen (22.9%) had a positive family history of stroke in first degree relative(s), 4 (6.5%) AD, and the family history was unknown for 9 (14.8%). We detected three APP mutations in three probands: c.2080G>A, p.Asp694Asn (known and hereafter referred as Iowa), c.2077G>A, p.Glu693Lys (Italian), and c.2075C>G, p.Ala692Gly (Flemish).
Case Fr-Iowa-1
A 58-year-old woman without any significant vascular risk factors except moderate smoking complained about word finding difficulties and memory impairment, which appeared progressively during the past two years. Neuropsychological assessment disclosed anomic aphasia with apathy and dysexecutive syndrome, a pattern suggestive of frontal subcortical dysfunction. CSF AD biomarkers analysis showed decreased Aβ42 levels (316 ng/L) and increased Tau (>1200 ng/L) and P-Tau (203 ng/L). CT-scan showed diffuse leukoaraiosis and cortical gyriform calcifications, located in the occipital cortex and in the depth of several sulci (Fig. 1A). On brain magnetic resonance imaging (MRI), calcifications were only suspected on susceptibility-weighted images (Fig. 1A). Severe leukoencephalopathy without cortical atrophy was noted, as well as 6 cortical or subcortical microbleeds. Hippocampi volumes were normal. At age 60, she experienced left frontal lobar hematoma(Fig. 1A).
Case Fr-Iowa-2
The 51-year-old brother of case Fr-Iowa-1 complained of progressive mild difficulties in counting and writing and a loss of driving abilities. He had a medical history of depression, one tonic-clonic seizure 7 years earlier that was attributed to alcohol use and benzodiazepine withdrawal, and acute pancreatitis. Neuropsychological assessment performed at 51 years disclosed marked impairment of verbal anterograde memory mainly related to a recall disorder, executive, attentional, and working memory disturbances. Visual perception, confrontation naming, and abstract thinking were preserved. MRI showed patchy confluent subcortical T2 hyperintensities (Fig. 1B). Susceptibility-weighted images were unavailable so that the presence of microbleeds could not be assessed but cortical occipital calcifications were seen on CT-scan (Fig. 1B).
Case Fr-Iowa-3
The father of cases 1 and 2 was born in Saxony and was of Austro-Hungarian descent. He had a medical history of meningitis at 46 years. When he was 47 years old, he suffered from his first episode of subarachnoid hemorrhage. Cerebral arteriography was normal. Clinical outcome was rapidly favorable. Four years later he suddenly presented with headache and aphasia, but his condition returned to normal in a few days. Lumbar puncture and cerebral arteriography were unremarkable (no AD biomarkers available). CT scan showed moderate subcortical and cortical atrophy and bilateral and symmetrical occipital cortical calcifications. When he was 56, he had a severe episode of headache with delirium. In the following years, he showed persistent disorientation, memory impairment, confabulations, mental slowing, and episodes of agitation. He died at 60 years.
Genetics of Fr-Iowa pedigree
We identified a c.2080G>A, p.Asp694Asn APP mutation in case Fr-Iowa-1. Her APOE genotype was 3/4. Her brother was then confirmed to be a carrier of the same APP mutation and of an APOE 3/3 genotype (Fig. 2A). DNA of their father is not available. This mutation was initially reported in two families [2, 4]. The first one originated from Iowa, and the kindred was of German descent [2]. As the pedigree of our patients indicated that they also originated from Germany, we hypothesized that they might have a common ancestor. We assessed six informative microsatellite-containing regions surrounding APP by PCR and found one allele shared between the index case of the original Iowa pedigree and both sib-pairs from the Fr-Iowa pedigree for 5 of the 6 microsatellites: two mapping centromeric from APP, two within APP intronic regions, and one telomeric from APP. The D21S1435 marker showed no allele in common between the two individuals and mapped even more telomeric. This is consistent with the hypothesis of a common ancestor.
Case Fr-Italian-1
A 56-year-old woman presented with transient visual disturbance of unidentified cause. MRI showed white matter hyperintensities. Her past medical history was marked by migraine without aura since the age of 30. She then presented with progressive memory impairment from the age of 60. On a control MRI at 61 years (Fig. 1C), microbleeds and sequelae of right cerebellar infarction were observed. At 65 years, she presented with right lobar occipital hematoma revealed by left homonymous lateral hemianopsia. Mini-Mental State Examination (MMSE) score was 24/30, and she presented with impaired episodic memory with inefficient cueing and preserved visual, confrontation naming, and executive functions. On T2* sequences and CT-scan, cortical occipital calcifications were observed (Fig. 1C). CSF biomarkers analysis showed decreased Aβ42 levels (334 ng/L) and increased Tau (422 ng/L) and P-Tau (77 ng/L). Her mother died at age 63 in the context of unspecified stroke.
Case Fr-Italian-2
The older brother of case Fr-Italian-1 presented with right occipital hematoma at age 60. Cerebral MRI revealed white matter hyperintensities and microbleeds, and CT scan also revealed cortical occipital calcifications. He was then lost tofollow up.
Genetics of Fr-Italian pedigree
We identified a c.2077G>A, p.Glu693Lys APP mutation in Case Fr-Italian-1. Her APOE genotype was 3/3. Her brother was then shown to be a carrier as well as of an APOE 3/3 genotype (Fig. 2B).
Case Fr-Flemish-1
A 47-year-old man of Portuguese origin presented with a two-year history of progressive memory loss and mild headache. He reported transient unilateral upper limb paresis at the age of 42. Neuropsychological assessment revealed a MMSE score of 19/30; Mattis Dementia Rating Scale score of 120/144, and impaired verbal episodic memory characterized by impaired encoding with inefficient cueing. Cerebral MRI revealed cortical and marked bilateral hippocampal atrophy, mild posterior leukoencephalopathy with lacunar strokes, and multiple cortical and juxtacortical microbleeds associated with one right thalamic hemorrhage and disseminated hemosiderosis on susceptibility-weighted images (Fig. 1D). An FDG-PET study revealed bilateral asymmetric parieto-temporal hypometabolism. No cortical calcifications were found on cerebral CT-scan (Fig. 1D). CSF biomarker analysis showed intermediate Aβ42 levels (594 ng/L) with normal Tau (226 ng/L), P-tau (45 ng/L), and Tau/Aβ42 ratio (0.38; N < 0.52).
His cognitively normal 37-year-old brother presented with a right thalamic hemorrhage with no history of high blood pressure. Cerebral MRI showed multiple cortical and subcortical microbleeds.
Their father died at 60 years after a 9-year history of progressive cognitive decline leading to a diagnosis of probable AD.
Genetics of Fr-Flemish pedigree
We identified a c.2075C>G, p.Ala692Gly APP mutation in Case Fr-Flemish-1. His APOE genotype was 3/3. DNA from his brother and father were not available (Fig. 2C).
Review of literature
The search yielded a total of n = 156 results, of which we retained 13 reports with phenotype information after manual curation. The results of this review are displayed in Supplementary Tables 1–3.
The APP Iowa mutation has previously been reported in only ten cases from five families [2–6, 9]. Added to our patients, a total of 10 genetically-ascertained cases and 11 affected relatives (5 obligate carriers) were considered (Supplementary Table 1). The mean age at clinical onset (among the 18/20 symptomatic patients with available information) was 51.3 years [34–67]. A total of 15/20 patients experienced lobar intracerebral hemorrhage (ICH), confirming that this type of event is a prominent feature associated with the APP Iowa mutation. Furthermore, microbleeds were observed on brain MRI in T2* images of one patient from the original Iowa family (no symptomatic lobar ICH) and were present prior to the lobar hematoma on susceptibility-weighted images in case Fr-Iowa 1.
Cortical calcifications were reported in 14 patients with an Iowa APP mutation. They involved occipital sulci in all of them, and were more rarely located in the depth of other sulci. For 5 obligate carriers from the Polish and Irish pedigrees and 2 presymptomatic carriers from the Polish pedigree (not displayed in the Supplementary Tables), CT-scans were either unavailable or were performed but the authors did not report the absence or the presence of calcification. Some of the available CT-scans might have been performed several decades ago, and faint cortical calcifications might have been missed, as is the case for basal ganglia calcifications on CT-scans of previous generation [10]. However, CT-scan did not show calcifications in 2/3 of the patients from the Spanish pedigree [9]. These scans were performed at 40 and 37 years, suggesting that this imaging feature could appear later in some patients.
The Italian APP mutation has previously been reported in 4 families from Southern Lombardy, Italy [11, 12]. The main clinical features in affected individuals were highly similar to the ones of patients carrying other APP mutations associated with CAA (in particular, Dutch and Iowa), i.e., recurrent hemorrhagic (lobar and subarachnoid) strokes and its related symptoms, including focal signs, cognitive impairment, and epilepsy (Supplementary Table 2). Lobar hematomas, unspecified stroke, or unspecified headache were noted in all 20 affected individuals from these families. Among the 14 patients with available information added to our 2 patients, the mean age at clinical onset was 54.1 years [44–63] [11]. Several of these patients underwent both CT-scan and MRI. In several of these MRI, cortical hypointensities on T2*-weighted images were reported. This can be consistent with either superficial siderosis/subarachnoid hemorrhages or with calcification. However, CT-scan is the sole sensitive and specific imaging tool allowing for the identification of calcifications. None were reported on theseCT-scans.
The APP Flemish mutation was previously reported in 19 patients (8 mutation carriers and 11 untested affected relatives) in the original family [13–15], 6 patients in another family (3 mutation carriers and 3 untested affected relatives) [16], and one additional patient [17]. For 15 patients (including ours), clinical description was provided: 13 were diagnosed with progressive dementia (definite AD, n = 4; possible/probable AD, n = 4, uncharacterized, n = 5) and 5 presented with at least one ICH, including 2 patients previously diagnosed with AD (Supplementary Table 3). Among 13 patients with available information, the mean age at clinical onset was 45.3 years [35–54]. No cortical calcification was reported in the literature as well as in our patient, who presented with probable CAA and a diagnosis of probable AD [18], which could not be confirmed biochemically. His brother’s history was positive for probable CAA (cortical and subcortical microbleeds), suggesting that he could be a carrier as well, but his DNA was not available. The presence of thalamic hemorrhage with no other cause in both brothers is atypical in this context.
DISCUSSION
We report three new families with CAA-associated APP mutations, two of which exhibited cortical calcifications. Cortical calcification was thought to be a highly specific imaging feature of the APP Iowa mutation in the context of CAA [2]. It was indeed found in all initially reported patients and no carrier of another APP mutation has previously been reported as exhibiting this feature. We report for the first time two patients with an Italian APP mutation and cortical calcifications. Such serpentine occipital calcifications can be observed in other disorders, which are typically associated with basal ganglia (and often other brain areas) calcifications. Among them, primary familial brain calcification due to SLC20A2, PDGFB, or XPR1 mutations [19, 20] and phosphocalcic metabolism disorders seem to be the most frequent causes in cases of adult presentations. Other rare diseases, such as Aicardi-Goutières syndrome, typically start during childhood [21]. In the absence of basal ganglia involvement, occipital cortical calcification has been reported in celiac disease with epilepsy and calcifications syndrome where calcifications appear with a coarser aspect than in other conditions [22], in very few patients with late onset sporadic CAA [4], and in several patients with transthyretin-related CAA [23, 24]. To our knowledge, no evidence of calcification was reported on CT-scans of other APP mutation carriers, although calcified CAA lesions were reported in 6 of 29 autopsied brains of patients with familial CAA due to an APP Dutch mutation [25] and in the parietal and occipital lobes of autopsied brains of patients with an APP duplication [26].
The mechanisms underlying vascular calcification in CAA are unknown and not specific to the Aβ peptide [23, 24]. Calcification might be the result of amyloid deposits in the vessel walls with severe amyloid charge, and its associated microvascular degeneration and inflammation. Why specific vascular beds would exhibit calcification or other secondary microvascular degeneration (hyalinization, microaneurysms, histiocytosis, lymphocytosis, granulomatous inflammation, vascular thrombi, or fibrinoid necrosis [25]) remains unanswered. Why calcification occurs specifically or more prominently in the occipital cortex also remains an open question. This seems to be consistent with the posterior predominance of amyloid deposits in CAA. However, the observation of calcifications in amyloid-free vessels might suggest alternative hypotheses [6]. Comparison with patients with an SLC20A2, PDGFB, or XPR1 mutation is interesting as, when cortical calcifications are observed, they are primarily located in the depth of sulci and/or the occipital cortex, with very similar images. Of note, one patient with transthyretin-related CAA exhibited insular calcifications [24]. It would also be interesting to screen CT scans from patients with other APP mutations, whatever the associated diagnosis of CAA or AD, to evaluate the specificity of cortical calcifications seen in the context of APP mutations.
Progressive cognitive decline was not associated with symptomatic lobar ICH in the original Iowa pedigree [2]. However, the autopsied patient IV.1 from the original Iowa family had small cortical hemorrhages and 15/20 APP Iowa mutation carriers eventually presented with lobar ICH. APOE ɛ4 allele was associated with an increased risk of sporadic CAA [27], while it has been suggested that APOE ɛ2 could be a risk factor for ICH in vessels with amyloid charge [28]. Whether the APOE genotype is a modifier of the clinical expression of APP mutations remains to be determined with larger samples sizes such as in the very large Colombian pedigree which allowed demonstrating that the APOE ɛ2 allele is a modifier of the age of onset of AD among the carriers of the PSEN1 p.E280A mutation [29].
The description of cerebral white matter was available from MRI results of 11 APP Iowa mutation carriers, including two from our sample. Nine showed diversely severe degrees of leukoencephalopathy and two had normal white matter (aged 53, otherwise affected by lobar ICH, and aged 30, presymptomatic carrier). No specific pattern was associated with CAA related to the APP Iowa mutation or the other two APP mutations.
Among APP mutation carriers, some patients experience progressive cognitive decline, with or without ICH, raising the question of co-occurrence of AD and CAA. Of note, no neurofibrillary tangles were observed in the sole patient with an Italian APP mutation and available neuropathological examination [11], whereas the previously autopsied patient with an Iowa APP mutation and detailed neuropathological information had numerous neurofibrillary tangles (Braak and Braak stage VI) but only some neuritic plaques [2]. The four autopsied Flemish APP mutation carriers were diagnosed with severe Aβ-CAA and definite AD [13, 30], with vasculocentric senile plaques with large cores predominantly composed of Aβ40 [30]. Although neuropathological data were not available in our pedigrees, we observed progressive cognitive decline in cases Fr-Iowa 1 and Fr-Italian 1 (in particular, amnestic syndrome with impaired episodic memory with inefficient cueing in Case Fr-Italian-1). This was associated with decreased CSF Aβ42 and increased Tau and P-tau. Non-demented patients with CAA exhibit decreased levels of CSF Aβ42, some of them presenting slightly increased Tau and P-tau, including one Iowa APP mutation carrier [31]. We can hypothesize that coexistence of such a pattern of CSF AD biomarkers with progressive cognitive decline might be the result of an AD pathophysiological process associated with Aβ-CAA. However, in the context of CAA, decreased Aβ42 levels may not be used as a marker of plaques. It would be interesting to know if the non-demented patients with CAA and decreased CSF Aβ42 and elevated Tau and P-tau levels [31] will exhibit progressive cognitive decline with neuritic plaques on neuropathological examination.
As this series is retrospective, independent prospective studies are needed to evaluate the mutational frequencies in patients fulfilling the same criteria. There is an increasing number of APP mutations reported in the context of early-onset AD and/or CAA. It would be of high interest to benefit from international registry for annual standardized neuroimaging and clinical follow-up to better describe the natural history of the disease and identify prognostic biomarkers as well as outputs for future clinical trials. Furthermore, a revision of the Boston criteria could be proposed to allow the inclusion of young patients.
In conclusion, we confirm that the p.Asp694Asn APP mutation, known as Iowa, results in severe CAA with cortical— mainly occipital— calcification and AD. Furthermore, we show for the first time that the p.Glu693Lys APP mutation, known as Italian, is responsible for a very similar phenotype, including occipital calcification. Although this imaging feature remains of important diagnostic value, its mechanisms and significance remain to be determined.
Footnotes
ACKNOWLEDGMENTS
We are indebted to all the neurologists who sent blood samples and medical charts.
This study was funded by grants from the Clinical Research Hospital Program from the French Ministry of Health (GMAJ, PHRC 2008/067) and the CNR-MAJ. The funding sources had no specific roles.
