Neurology. 2015 Jun 2;84(22):2266-73.

Homozygous carriers of APP A713T mutation in an autosomal dominant Alzheimer disease family.

 

M.E. Conidi1*(PhD), L. Bernardi1* (PhD), G. Puccio1 (MD), N. Smirne1 (BS), M.G. Muraca1 (MRS), S.A.M. Curcio1 (PhD), R. Colao1 (MD), P. Piscopo2 (PhD), M. Gallo1 (PhD), M. Anfossi1 (PhD), F. Frangipane1 (MD), A. Clodomiro1. (MD), M. Mirabelli1 (PhD), F. Vasso1 (TC), C. Cupidi1 (MD), G. Torchia1 (BS), R. Di Lorenzo1 (MD), P. Mandich3 (MD), A. Confaloni2 (PhD), R.G. Maletta1 (MD) and A.C. Bruni1§.(MD).

  1. Regional Neurogenetic Centre, ASP Catanzaro, Lamezia Terme (CZ) Italy
  2. Department of Cell Biology and Neurosciences, National Institute of Health, Rome, Italy
  3. DINOGMI – Università degli studi di Genova

* These authors contributed equally to this work

 

ABSTRACT

Objective: To report, for the first time, a large autosomal dominant Alzheimer disease (AD) family in which the APPA713T mutation is present in the homozygous and heterozygous state. To date, the mutation has been reported as dominant, and in the heterozygous state associated with familial AD and cerebrovascular lesions. Methods: The family described here has been genealogically reconstructed over 6 generations dating back to the 19th century. Plasma b-amyloid peptide was measured. Sequencing of causative AD genes was performed.

Results: Twenty-one individuals, born from 2 consanguineous unions, were recognized as affected by dementia: 8 were described as affected through history, 5 were studied clinically and genetically, and 8 were asymptomatic at-risk subjects. The A713T mutation was detected in the homozygous state in 3 patients and in the heterozygous state in 8 subjects (6 asymptomatic and 2 affected).

Conclusions: Our findings, also supported by the beta-amyloid plasma assay, confirm (1) the pathogenic role of the APP A713T mutation, (2) the specific phenotype (AD with cerebrovascular lesions) associated with this mutation, and (3) the large span of age at onset, not influenced by APOE, TOMM40, and TREM2 genes. No substantial differences concerning clinical phenotype were evidenced between heterozygous and homozygous patients, in line with the classic definition of dominance. Therefore, in this study, AD followed the classic definition of a dominant disease, contrary to that reported in a previously described AD family with recessive APP mutation. This confirms that genetic AD may be considered a disease with dominant and recessive traits of inheritance.

PMID: 25948718

 

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INTRODUCTION

Heterozygous mutations in the amyloid precursor protein (APP) gene are associated with different phenotypes such as autosomal dominant early-onset Alzheimer’s disease (ADEOAD) (1,2,3), cerebral amyloid angiopathy (CAA) (4), ADEOAD with CAA (5) and hereditary cerebral hemorrhage with amyloidosis (6). Two recessive mutations, causing disease in the homozygous state, have also been identified (7,8) in two different families. Heterozygous APP mutations affecting codons from 714 to 717 alter γ-secretase cleavage causing an increase of the Aβ42/Aβ40 ratio (9,10). In AD families, Aβ42 elevated levels have been reported among symptomatic carriers of presenilin mutations (11), and Aβ42 and Aβ42/Aβ40 ratio levels were higher in unaffected familial AD mutation carriers compared with the unaffected non mutated individuals belonging to the same AD families (12). First degree relatives of patients with late onset AD (LOAD) without any known mutations have also been found to have an increase of plasma Aβ42 (13). The APP A713T mutation has been reported with dominant inheritance and in heterozygosis, associated with familial AD with both early and late onset and cerebrovascular lesions (CVLs) (5,14,15). All Italian A713T mutated patients (5,15) shared a common DNA haplotype, suggesting that this mutation originated from a putative ancestor (16), probably having lived centuries ago in Calabria (Southern Italy), where a high prevalence of this mutation has been observed (16).

In our study we report a large family (named PEC family) whose pedigree has been reconstructed over six generations from the present time up to the 19th century (subject II-2 was born in the year 1823) through the systematic collection of data from Municipal documents (births, deaths and marriage acts since 1809). Forty-nine people were identified; among them, 21 subjects (13 males, 8 females), over the three last generations were identified as at risk or affected by dementia. Twenty of these 21 individuals were born from two consanguineous unions. Eight persons were reported to be affected by dementia through history (5 males, 3 females), five were studied clinically and genetically (5 males, average age at onset 70.2±5.2 years, range 62-76 years). Eight asymptomatic at risk subjects (3 males, 5 females, average current age 56.4±7.3 years, range 47-66) were also clinically and genetically investigated. Genetic screening was conducted in all the available family members (5 affected and 8 asymptomatic at-risk subjects) for AD genes.

Sequencing of the APP gene exon 17 revealed the previously reported A713T mutation (275329 G>A, Genebank accession number D87675.1) (5,14) in 11 subjects (6 asymptomatic and 5 affected) and was excluded in 2 at risk relatives (asymptomatic subjects VI-7 and VI-8, females, current age, 57 and 63 years) (Tab.1).

 

Table 1. Demographic, clinical, genetic and biochemical data of family members;

Subject Gender Diagnosis Current age Onset age APP A713T mutation state Disease duration First sign at onset Cognitive impairment/ Behavioral Disorder Plasma Aβ42 (pM) Plasma Aβ40 (pM)
(V-2) M FAD Deceased at 84 years 76 Homozygous 12 Memory loss +/- NA NA
(V-17) M FAD  

84

70 Homozygous 14 Memory loss +/Apathy 10,4 232,0
(V-10) M FAD  

75

70 Homozygous 5 Memory loss +/Apathy

aggressiveness

15,8 381,2
(V-5) M FAD  

89

62 Heterozygous. 27 Memory loss +/Irritability, emotional flattening (after 13 yrs of onset) 9,4 265,9
(V-7) M FAD 77 73 Heterozygous. 4 Memory loss +/- 10,6 274,0
(VI-5) F Cognitively healthy 66 / Heterozygous 12,0 191,3
(VI-9) F Cognitively healthy 47 / Heterozygous. 12,6 189,5
(VI-1) M Cognitively healthy 64 / Heterozygous. 9,4 157,3
(VI-11) M Cognitively healthy 50 / Heterozygous 8,0 203,0
(VI-6) F Cognitively healthy 54 / Heterozygous. 6,2 149,6
(VI-10) M Cognitively healthy 50 / Heterozygous. 9,4 178,7
(VI-7) F Cognitively Healthy 57 / No mutation carrier 6,2 263,0
(VI-8) F Cognitively Healthy 63 / No mutation carrier 6,2 216,0

NA= not available

 

 

The mutation was found in the homozygous state in 3 patients (patients V-2, V-10, V-17, males, onset age 76, 70 and 70 years, respectively), heterozygous in 2 patients (patients V-5 and V-7, males, onset age 62 and 73 years, respectively) and in 6 asymptomatic at risk individuals (3 males, 3 females, average current age 55.2 ± 8.0 years, range 47-66 years) (Tab.1). In silico analyses predicted that the mutation is functionally “probably damaging” (PolyPhen-2 score of 1.000), “damaging” (SIFT score of 0.00) and “disease causing” (MutationTaster, probability value close to 1 indicates a high ‘security’ of the prediction), respectively.

The APP variation was absent in the control group, constituted by 800 chromosomes, belonging to Calabrian population.

We also collected plasma samples from 12 available family members, of these 10 were mutation carriers (2 homozygotes and 8 heterozygotes), and 2 were not mutated and born from a heterozygous mutation carrier. Each plasma sample was assayed for the Aβ peptide dosage to evaluate the effect of this mutation on the Aβ42/Aβ40 ratio.

Genetic results

We report here, to our knowledge for the first time, three individuals, affected by autosomal dominant AD, carrying the APP A713T mutation in the homozygous state. They were born to consanguineous parents and belong to a pedigree that also includes two affected and six at risk heterozygous carriers of the same mutation. The genealogical reconstruction showed that all the homozygous patients were born from consanguineous parents (second cousins, IV-1 and IV-2; first cousins IV-3 and IV-4); it is clear, indeed, that both parents of the three homozygous patients were obligate mutation carriers. On these bases, the homozygosity of the three patients was unquestionably verified.

We confirmed that the APP A713T variant is a mutation (and not a common polymorphism), given that it was absent in a control group constituted of 400 subjects belonging to the Calabrian population. On the other hand, the control sample size was not sufficiently large enough to exclude the variation as a very rare polymorphism.

The APP A713T is likely to be a functional variant with a probable deleterious effect because in silico analyses predicted that the mutation is functionally “damaging” and “disease causing”.

Furthermore, in this family, segregation of the mutation with disease is disclosed, given that all affected subjects carry the mutation in homozygous or in heterozygous state and given that heterozygous asymptomatic subjects are younger than the average onset of patients, thus they cannot be considered “escapees”.

Biochemical results

Comparing our data with findings from other APP mutations, A713T results in a simultaneous decrease of Aβ40 and increase of Aβ42 plasma levels in asymptomatic mutation carriers (ASMCs), similarly to that observed for the pathogenic APP T714I mutation (9), in line with evidence that mutations at codon 714 appear to primarily affect γ40-cleavage and cause a decreased secretion of Aβ40 (10). Our data from plasma show that the Aβ42/Aβ40 ratio increased in ASMCs with respect to non-mutation carriers (NMCs), as previously reported (12), and then it decreases in affected (homozygotes + heterozygotes) mutation carriers (AMCs) (Fig.1).

 

 

EC FIG1

Figure 1. The plot indicates mean Aβ42/Aβ40 ratio according to genotype in non-mutation carriers (NMCs), asymptomatic heterozygous mutation carriers (ASMCs) and affected mutation carriers (AMCs) belonging to the family.

 

The Aβ42/Aβ40 ratio resulted ~2-fold higher in the group of AMCs (p < 0.05) compared to the group of NMCs, as previously demonstrated in one AD patient carrier of the APP I716V mutation (17). These observations are consistent with findings published in other longitudinal studies, suggesting that plasma levels of Aβ peptides increase in the preclinical phase of AD, successively declining with the progression of clinical dementia, reflecting the deposition of Aβ peptides in the brain (18-23).

Moreover, homozygous and heterozygous AMCs had no significant difference in plasma levels of Aβ40, Aβ42, or Aβ42/Aβ40 ratio, thus showing the lack of “dose effect” of the mutation in plasma levels of Aβ peptides. This evidence is consistence with the fact that no substantial differences concerning clinical phenotype and course were evidenced comparing heterozygous and homozygous patients (Tab.1). In these affected persons, homozygosity for the APP A713T mutation does not aggravate the clinical picture of the disease, which is in line with the classical definition of dominance. Of note, the very long course in both genotypes.

Our study presents some limitations in terms of the biochemical results in plasma: 1) it is possible that the small number of the APP A713T mutation carriers could have influenced the significance of the data, despite these mutated subjects are very few in the world; 2) the NMCs subjects in this study included only two persons, given that we chose the available unaffected non-mutated individuals belonging to present family; 3) in this study it was not possible to perform serial measurements of Aβ peptides prior to the onset and during disease progression, as recommended in ADAD families with asymptomatic mutation carriers. Indeed, the diagnostic role of plasma as biomarker in these subjects remains to be further verified; we were unable for several reasons to measure the Aβ42/Aβ40 ratio in the CSF, largely recognized as the true biomarker (24-26).

 

The importance of this study

First, we report, for the first time, a large autosomal dominant AD family in which a mutation in a causative gene is present in the homozygous and heterozygous state. Our findings confirm the pathogenic role of the APP A713T mutation and the large span of onset, already reported to range from 52 to 82 years (5,15) and not influenced by APOE, TOMM40 rs10524523, and TREM2 rs75932628 genotype, reported significantly associated to the risk of AD (27,28) (Tab.1). Probably, this large span of onset and disease course may be due to a specific genetic background of modifier genes and/or to epigenetic effect, that in the future would be interesting to investigate.

The phenotype of the PEC family, associated with this mutation, and showing AD with CVLs (evident at neuroimaging, but without neurological signs), is not different from the clinical picture observed in other APPA713T affected subjects we previously described, probably being a distinctive feature, at least in the Calabrian population (16). Although neither in-depth genealogical reconstruction nor haplotype analysis were carried out in the APP mutation carriers belonging to the different APPA713T calabrian families, we hypothesize that they are related and probably originated from the same common ancestor prior to the 19th century (16).

In this study, AD behaves as a real dominant disease, similar to that reported for the rare homozygous CADASIL patients (29, 30) and differently from that reported in the AD family with the recessive APP A673V mutation (7) that causes disease only in the homozygous state, whereas heterozygous carriers were unaffected, consistent with a recessive trait of inheritance. All these data confirm that genetic AD may be considered a disease with dominant and recessive traits of inheritance, thus demonstrating the importance to search for recessive AD loci, for instance by analyzing homozygosity in consanguineous families and inbred populations.

Second, we observed that APP A713T increases the Aβ42/Aβ40 ratio in plasma (31). Studies in cell culture may elucidate the probable deleterious effects of the mutation on APP processing, and whether the presence of genetic risk factors relevant in other neurodegenerative dementias may affect this.

For reasons that are currently unclear, A713T APP mutation was identified in patients either with classic Alzheimer’s pathology or prominent CAA with significant vascular lesions. It would be interesting to determine the pattern of amyloid pathology in a mouse model and to explore potential modifiers of that pathology.

Further NGS studies of this family may allow us to highlight the role of rare susceptibility modifying alleles or the putative involvement of new genes in AD genetics and the key role of Aβ network alteration in AD.

 

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Contact:

Dr. A.C. Bruni, Centro Regionale di Neurogenetica, Azienda Sanitaria Provinciale Catanzaro, Viale A. Perugini, 88046 Lamezia Terme (CZ) Italy, Tel.:+39-0968 208080, Fax:+39-0968 208032, e-mail bruni@arn.it

 

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