- Email: [email protected]
Unrecognized Fibrinogen A α-Chain Amyloidosis: Results From Targeted Genetic Testing
Original Investigation Unrecognized Fibrinogen A a-Chain Amyloidosis: Results From Targeted Genetic Testing Isabel Tavares, MD,1,2 Joa˜o Paulo Oliveira, MD, PhD,2,3 Ana Pinho, MD,1 Luciana Moreira, PhD,4 Liliana Rocha, MSc,3 Josefina Santos, MD,5,6,7 Joaquim Pinheiro, MD, PhD,8 Paulo Pinho Costa, MD, PhD,4,6 and Luı´sa Lobato, MD, PhD 5,6 Background: Fibrinogen A a-chain (AFib) amyloidosis results from autosomal-dominant mutations in the gene encoding AFib (FGA). Patients with this disorder typically present with proteinuria. Isolated cases of AFib amyloidosis, carrying the FGA p.Glu545Val variant, were identified in the district of Braga, in northwest Portugal. This observation led us to hypothesize that this disorder might be an unrecognized cause of kidney disease in that region and prompted us to carry out targeted genetic testing for the p.Glu545Val variant in the local hemodialysis population and family members of identified cases. Study Design: Case series. Setting & Participants: 3 groups of participants: (1) kidney biopsy registry, n 5 4; (2) hemodialysis facility, n 5 122 of 267 patients; and (3) genetically at-risk individuals; n 5 69 of 167 family members. Outcomes: Kidney disease, kidney disease progression, and survival. Results: The p.Glu545Val variant was identified in all 4 patients of the biopsy registry, 12 of 122 (9.8%) hemodialysis patients tested, and 34 of 69 (49%) relatives tested. These 50 cases belonged to 13 unrelated families with kidney disease or amyloidosis identified in 61% of probands. 35 individuals presented with hypertension at a mean of 51.0 6 10.4 years. Of these, 30 developed kidney disease at a mean of 56.7 6 12.0 years, and 21 initiated dialysis therapy at a mean of 61.4 6 11.3 years. Heart, liver, spleen, colon, and ileum were involved along the progression of the disease. Kidney disease was formerly attributed to hypertension in 25% of patients with AFib amyloidosis undergoing hemodialysis. Limitations: Retrospective data collection for patients with amyloidosis previously diagnosed. Conclusions: AFib amyloidosis appears to be an under-recognized disorder in Braga, Portugal, where we found a high frequency of the FGA p.Glu545Val variant. Due to the nonspecific nature of its major clinical features, the diagnosis of AFib amyloidosis should have a high index of suspicion, particularly in populations in which hypertension is prevalent. Am J Kidney Dis. 70(2):235-243. ª 2017 by the National Kidney Foundation, Inc. INDEX WORDS: Amyloidosis; chronic kidney disease (CKD); fibrinogen A alpha-chain; genetic screening; hemodialysis; mutation; proteinuria; hypertension; FGA p.Glu545Val; Portugal; end-stage renal disease (ESRD).
F
ibrinogen A a-chain (AFib) amyloidosis is a systemic disease caused by extracellular deposition of insoluble amyloid fibrils composed of abnormal fibrinogen, arising from autosomal-dominant mutations in the gene encoding AFib (FGA).1-5 Patients with AFib amyloidosis invariably develop chronic kidney disease (CKD), typically progressing to end-stage renal disease (ESRD) within 5 years of recognition of renal involvement.3 Diagnosis is based on the occurrence of proteinuric nephropathy, positive family history, identification of amyloid deposits in affected tissues From the 1Department of Nephrology, Centro Hospitalar de São João; 2Group of Research and Development in Nephrology and Infectious Diseases, Institute of Biomedical Engineering–I3S, and 3 Department of Genetics, Faculty of Medicine, University of Porto; 4 Department of Human Genetics, National Health Institute Doutor Ricardo Jorge; 5Department of Nephrology, Centro Hospitalar do Porto, Hospital de Santo António; 6Unit for Multidisciplinary Research in Biomedicine, Instituto de Ciências Biomédicas Abel Salazar, University of Porto, Porto; 7Dialysis Clinic of Braga, NephroCare–Portugal, Braga; and 8Dialysis Clinic of Fafe, NephroCare–Portugal, Fafe, Portugal. Am J Kidney Dis. 2017;70(2):235-243
by immunohistochemistry or mass spectrometry, and detection of an FGA amyloidogenic genetic variant.1-6 Incomplete penetrance may complicate the diagnosis of AFib amyloidosis and should be taken into account in genetic counseling. To date, 13 amyloidogenic FGA variants have been described,7 accounting for 8% of hereditary amyloidoses.8 Although AFib amyloidosis was originally described in 1993 in a Peruvian kindred, segregating with a mutation in FGA identified as p.Arg554Leu (ie, indicating a substitution of arginine by leucine at amino Received May 18, 2016. Accepted in revised form January 3, 2017. Originally published online March 27, 2017. Address correspondence to Isabel Tavares, MD, Department of Nephrology, Centro Hospitalar de São João, Alameda Professor Hernâni Monteiro, 4200-319 Porto, Portugal. E-mail: isabel. [email protected] 2017 by the National Kidney Foundation, Inc. 0272-6386 http://dx.doi.org/10.1053/j.ajkd.2017.01.048
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acid 554),1 the most common FGA amyloidogenic variant reported worldwide is one described as p.Glu526Val (substitution of glutamate by valine at amino acid 526), having been identified in a Canadian kindred of Polish origin9 and in families from the United Kingdom,3,10 France,11 Germany,12 Brazil,13 United States,14 and China.15 According to recommendations of the Human Genome Variation Society,16 the p.Arg554Leu and p.Glu526Val variants should be described, respectively, as p.Arg573Leu and p.Glu545Val (ie, considering the translation initiation site as amino acid 1, instead of basing the numbering on the cleaved protein). In line with this, the Nomenclature Committee of the International Society of Amyloidosis17 recommends that hereditary amyloidosis associated with the FGA p.Glu545Val variant should be designated AFibE526V (p.Glu545Val) amyloidosis, and this nomenclature will be followed hereinafter. In Portugal, the first patient with AFib amyloidosis was reported in 2004,2 and the p.Glu545Val variant was found to be the causative mutation. Subsequently, 4 other apparently unrelated patients,18 including a woman with a homozygous mutation,19 were given a diagnosis of AFib amyloidosis by retrospective immunohistochemical characterization of 102 kidney
biopsies showing amyloid nephropathy, retrieved from the archive of Centro Hospitalar de São João, a major university hospital in Porto, in the northwest of Portugal. Because all 4 patients carried the p.Glu545Val variant and were from the same confined geographic area, we hypothesized that AFibE526V (p.Glu545Val) amyloidosis may be an underdiagnosed cause of ESRD in that region and carried out targeted genetic screening among the local outpatient hemodialysis population, offering cascade genetic screening to at-risk relatives of all identified cases. We report demographic and clinical features of individuals carrying the FGA p.Glu545Val variant identified in this study, irrespective of their medical condition, and characterize the natural history and major outcomes of AFibE526V (p.Glu545Val) amyloidosis.
METHODS Study Design, Participant Ascertainment, and Data Collection Participants enrolled in this study were ascertained by 3 distinct sequential case-finding protocols (Fig 1), starting with the 4 patients who were retrospectively given diagnoses of AFibE526V (p.Glu545Val) amyloidosis, by review and immunohistochemical classification of archived kidney biopsies showing amyloid nephropathy (phase I).18
Phase I Review of the kidney biopsy registry of a major university hospital [1977-2013]: N=3,264
Histological diagnosis of amyloid nephropathy N=102
Immunohistochemical diagnosis of amyloid nephropathy due to AFib amyloid fibril protein N=4
Phase II
Phase III
Genetic diagnosis of AFib amyloidosis due to the FGA p.Glu545Val variant N=4
(all patients from a confined region in the district of Braga, NW Portugal)
Review of the prevalent patient databases of 3 local outpatient hemodialysis clinics [2005-2010]: N=267
13 unrelated families diagnosed with AFib amyloid nephropathy (2005-2014) FGA p.Glu545Val N=12 At-risk relatives N=167
Accepted genetic screening N=69
Accepted genetic screening N=122
Non-stringently defined or undetermined ESRD etiology or unclassified amyloid nephropathy N=122
Exclusion criteria: • etiology of ESRD stringently defined: N=126 • no informed consent: N=19
FGA p.Glu545Val N=34
Figure 1. Study design shows setting, eligibility, and results of included cases. Targeted genetic testing led to the identification of 50 individuals with the FGA p.Glu545Val variant in the same geographical region. Abbreviations: AFib, fibrinogen A a-chain; ESRD, end-stage renal disease; FGA, fibrinogen A a-chain gene; NW, northwest. 236
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Unrecognized Fibrinogen A a-Chain Amyloidosis The observation that the families of all 4 probands originated from neighboring villages in the district of Braga prompted a casefinding study among the local regular hemodialysis population (phase II). To this end, 267 prevalent patients with ESRD, 18 years or older, and undergoing hemodialysis in 3 outpatient clinics in the district of Braga between January 2005 and June 2010 were evaluated for eligibility for genetic screening. Criteria for offering p.Glu545Val genotyping included CKD of undetermined or nonstringently defined cause (diabetic nephropathy in the absence of diabetic retinopathy, hypertensive nephrosclerosis, and glomerular and interstitial diseases in the absence of histologic confirmation) or a diagnosis of unclassified amyloid nephropathy. Exclusion criteria were a stringently defined CKD diagnosis, either biopsy proven or inferred from a highly specific clinical context, or refusal or inability to provide informed consent. When a patient carrying the p.Glu545Val variant was identified, the relevant demographic and clinical data were collected, including age at onset of clinical manifestations; renal laboratory data, and age at the initiation of renal replacement therapy; history of vascular, gastrointestinal, and neurologic morbidity and their respective clinical outcomes; and age at death. Additional clinical data were retrospectively collected by review of archived medical records, the oldest available dating as far back as 1973, or by interviewing the patient’s relatives. A detailed genogram capturing at least 3 generations was drawn for every patient diagnosed in phases I and II to check for possible relatedness among their families, as well as to identify genetically at-risk individuals. The latter were offered genetic counseling and those who eventually accepted genetic testing (phase III) underwent comprehensive baseline clinical assessment, including repeat blood pressure readings, plasma creatinine and proteinuria measurements, and kidney ultrasonography. Follow-up specialized medical care was offered to all participants in this study, and the relevant clinical events in their natural histories were prospectively collected until December 2014. For the purpose of this study, the diagnosis of AFibE526V (p.Glu545Val) nephropathic amyloidosis did not require histologic confirmation of amyloid deposition on a kidney biopsy; patients harboring the p.Glu545Val variant were regarded as affected if they also had CKD, as defined in the next paragraph, whereas those who did not display significant manifestations of renal involvement were designated as genetic carriers. Furthermore, FGA genotyping was a requisite for enrollment in this study of the genetically at-risk individuals identified by pedigree analysis, irrespective of medical history or current clinical condition. Criteria for diagnosis of CKD were proteinuria with protein excretion $ 0.15 g/24 h or estimated glomerular filtration rate (eGFR) , 60 mL/min/1.73 m2 for 3 or more months, as calculated by the CKD-EPI (CKD Epidemiology Collaboration) creatinine equation.20 Staging of CKD was according to the KDIGO (Kidney Disease: Improving Global Outcomes) guideline.21 Systemic hypertension was defined as systolic blood pressure $ 140 mm Hg and/or diastolic blood pressure $ 90 mm Hg or current antihypertensive drug treatment. The clinical diagnosis of hypertension was based on a blood pressure assessment made at the time of blood sampling for FGA genotyping, as well as on historical records and/or prospective measurements obtained within 1 year of the baseline evaluation. In the absence of CKD, hypertensive patients carrying the p.Glu545Val variant were not given a diagnosis of nephropathic amyloidosis but instead were classified as genetic carriers. This study was conducted in accordance with the Ethical Principles for Medical Research Involving Human Subjects adopted by the World Medical Association,22 and its design and research protocol were reviewed and approved by the Centro Hospitalar de São João Health Ethics Commission (resolution no. 90/2012). Written informed consent was obtained from all participants. Am J Kidney Dis. 2017;70(2):235-243
Molecular Analysis Genomic DNA was extracted from peripheral-blood samples with the Prepito DNA Blood250 Kit (Perkin Elmer) according to the manufacturer’s instructions. The p.Glu545Val variant, coded for by c.1634A.T (an adenine to thymidine substitution at nucleotide 1,634 of the coding sequence, in exon 5 of the FGA gene) was screened by polymerase chain reaction amplification followed by Sanger sequencing using forward primer 50 -CTTCATCTGCAGTCAGCGATC-30 and reverse primer 50 -GAGATTTAGCATGGCCTCTC-30 . Polymerase chain reaction amplification was carried out in a Veriti thermal cycler (Applied Biosystems) with the following conditions: 95 C for 5 minutes, 35 amplification cycles (of denaturation at 95 C for 1 minute, annealing at 61 C for 1 minute, and extension at 72 C for 1 minute), and terminal extension at 72 C for 10 minutes. Polymerase chain reaction products were analyzed with the QIAXcel system (QIAGEN), purified with Agencourt AMPure XP–PCR Purification (Beckman Coulter), and sequenced on an automated ABI PRISM TM 3500 Genetic Analyser (Applied Biosystems) using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The National Center for Biotechnology Information reference sequences NM_00050823 and NP_00049924 were used as the references for the FGA complementary DNA and protein sequences, respectively.
Statistical Analyses Categorical variables were expressed as number and percentage; continuous variables were expressed as mean with standard deviation or as median with interquartile range, depending on whether they were normally distributed. Individuals carrying the p.Glu545Val variant were grouped according to the case-finding protocol in which they were identified, and the 3 cohorts were compared for relevant demographic and clinical data using Fisher exact test for categorical variables and 1-way analysis of variance or Kruskal-Wallis test for continuous variables, depending on goodness of fit to normal distribution. Prevalence, age of onset, and severity of hypertension were compared in p.Glu545Val carrier and noncarrier siblings, prospectively identified in family screening. To minimize ascertainment bias, family probands (n 5 10) were excluded from these analyses. The t test or c2 test, respectively, was used for comparing normally distributed continuous variables or categorical variables. The relationship between proteinuria and eGFR in the 30 patients with CKD was analyzed by Pearson correlation coefficient, using the first available proteinuria value with protein excretion $ 0.15 g/24 h. Decline in eGFR over time was analyzed by mixed-effects model in the 20 patients with CKD who had at least 2 serum creatinine measurements obtained more than 1 year apart. Absolute risks for major adverse outcomes (ie, ESRD and death) were estimated for the entire cohort (n 5 50), using Kaplan-Meier curves to determine renal and overall mean survival, censoring for death in the case of ESRD. P , 0.05 was considered statistically significant. All statistical analyses were performed using the IBM SPSS, version 20.0, software package for Macintosh.
RESULTS Study Population As shown in Fig 1, a total of 195 individuals eventually underwent genetic testing for the FGA p.Glu545Val variant, including the 4 patients retrospectively given diagnoses of AFib amyloidosis in phase I; all the eligible 122 hemodialysis patients enrolled in phase II; and 69 of 167 (41.3%) first-degree relatives of patients with AFibE526V (p.Glu545Val) amyloidosis who were identified in the 2 case-finding 237
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protocols. All those individuals were of Portuguese ancestry, with family roots in the district of Braga. The diagnosis of AFibE526V (p.Glu545Val) amyloidosis was established by molecular analysis in all 4 patients enrolled in phase I and in 12 of 122 (9.8%) hemodialysis patients screened in phase II. Of the latter patients, 6 were given diagnoses of unclassified amyloid nephropathy, whereas 3 had received a diagnosis of hypertensive nephrosclerosis and the remaining 3 had been classified as having ESRD of undetermined cause. On family screening, 34 of 69 (49%) individuals were found to be heterozygous for the p.Glu545Val variant, including 14 (41%) patients with evidence of CKD, none of whom was unaware of their clinical condition. Overall, the p.Glu545Val variant was identified in 50 individuals from 13 apparently unrelated families. Notably, 7 individuals from 3 unrelated families had died while receiving regular hemodialysis treatment before the beginning of this study and therefore did not have a definite genetic diagnosis. Characteristics of Individuals Carrying the FGA p.Glu545Val Variant
summarized for the overall cohort and for participants grouped according to the case-finding protocol in which they were identified. Mean age at genetic testing was 52.0 6 16.8 years. Hypertension was detected in 35 (70%) individuals at a mean age of 51.0 6 10.4 years and was present even in the absence of kidney disease. Thirty (60%) individuals developed CKD at a mean age of 56.7 6 12.0 years, presenting with proteinuria with protein excretion . 3 g/24 h and eGFR , 60 mL/min/1.73 m2 in more than half the cases, but only 17 (57%) such patients had undergone a diagnostic kidney biopsy. In phase III, a total of 20 of 34 (59%) individuals with the p.Glu545Val variant had no evidence of CKD at a mean age of 38.4 6 9.7 years and were therefore classified as genetic carriers. These individuals were significantly younger than their 14 relatives with CKD diagnosed, with a mean age difference of 18.4 years. Five (25%) individuals with no evidence of CKD had high blood pressure diagnosed as essential hypertension. Kidney Disease and Other Major Clinical Outcomes
The relevant demographic and clinical characteristics of individuals with the p.Glu545Val variant diagnosed in this study, irrespective of clinical condition, are presented in Table 1. The data are
All individuals with CKD presented with proteinuria. As shown in Fig 2, the severity of proteinuria associated inversely with eGFR (r 5 20.426; P 5 0.02), and mean change in eGFR was 24.39
Table 1. Diagnostic Assessment and Main Clinical Characteristics of Individuals With the FGA p.Glu545Val Variant Ascertainment Overall (N 5 50)
Kidney Biopsy Registry (n 5 4)a
Hemodialysis Screening (n 5 12)a
Family Screening (n 5 34)
P
Male sex Age at genetic testing, y Hypertension Age at diagnosis, y
22 (44) 52.0 6 16.8 35 (70) 51.0 6 10.4
2 (50) 57.5 6 16.6 4 (100) 52.8 6 12.5
6 (50) 67.2 6 12.2 12 (100) 52.6 6 11.8
14 (41) 46.0 6 14.7 19 (56) 49.7 6 9.5
0.8 ,0.001 0.006 0.7
Kidney disease presentation Age at presentation, y Proteinuria ,1 g/24 h 1-3 g/24 h .3 g/24 h eGFR ,60 mL/min/1.73 m2 $60 mL/min/1.73 m2
30 (60) 56.7 6 12.0
4 (100) 53.0 6 12.5
12 (100) 59.7 6 10.9
14 (41) 55.2 6 13.1
,0.001 0.5 0.05
9 (30) 4 (13) 17 (57)
0 (0) 1 (25) 3 (75)
1 (8) 2 (17) 9 (75)
8 (57) 1 (7) 5 (36)
19 (63) 11 (37)
3 (75) 1 (25)
12 (100) 0 (0)
4 (29) 10 (71)
Renal replacement therapy Age at initiation, y Dialysis vintage, mob Kidney transplantation
21 (42) 61.4 6 11.3 48.0 [27.0-90.5] 2 (10)
4 (100) 55.0 6 12.6 13.0 [3.8-203.0] 1 (25)
12 (100) 64.8 6 11.2 77.5 [47.2-91.2] 1 (8)
5 (15) 58.2 6 9.0 35.0 [18.5-67.0] 0 (0)
0.02
,0.001 0.3 0.1 NAc
Note: Values for categorical variables are given as number (percentage); values for continuous variables, as mean 6 standard deviation if normally distributed or median [interquartile range] if non-normally distributed. P values are from Fisher exact test for categorical variables and 1-way analysis of variance or Kruskal-Wallis for continuous variables, as appropriate. Abbreviations: eGFR, estimated glomerular filtration rate; NA, not applicable. a Patients ascertained by kidney biopsy registry and hemodialysis screening belonged to 13 unrelated families. b End of follow-up December 2014. c No applicability in this category. 238
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A
B
eGFR slope = - 4.39 mL/min/1.73m2
r= - 0.426
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Figure 2. (A) Scatterplot of proteinuria levels by estimated glomerular filtration rate (eGFR) for the 30 individuals with chronic kidney disease ascertained through a kidney biopsy registry (n 5 4), hemodialysis patient screening (n 5 12), and cascade family screening (n 5 14), analyzed by Pearson correlation coefficient (r 5 20.426; P 5 0.02). (B) eGFR regression slope for 20 of the 30 patients with chronic kidney disease who had at least 2 serum creatinine measurements available for review.
n 5 1; and spleen, n 5 1). Fourteen patients have died, at a mean age of 70.8 6 9.6 years, and their reported immediate causes of death are also summarized in Table S1. For the 50 patients with the p.Glu545Val variant (Fig 3), estimated mean renal survival was 65.4 (95% CI, 61.8-69.0) years, and estimated mean lifetime survival was 73.7 (95% CI, 70.5-76.8) years.
(95% confidence interval [CI], 23.65 to 25.14) mL/ min/1.73 m2 per year. Ten men and 11 women progressed to ESRD, having initiated renal replacement therapy at 61.4 6 11.3 years (Table 1); 2 women underwent kidney transplantation 6 years after the onset of hemodialysis therapy. Family history of kidney disease or amyloidosis was present in 61% of probands. Table S1 (provided as online supplementary material) summarizes the most relevant comorbid conditions and severe medical events that have occurred in the 30 patients with CKD. Thirteen strokes were reported (6 events in 6 women and 7 events in 5 men), with the middle cerebral artery territory being the most commonly affected location. Mean age for first stroke was 66.4 6 8.3 years. Amyloid deposits were also identified in surgical samples from hemodialysis patients (abdominal fat, n 5 1; colon, n 5 1; ileum, A
Survival Function Censored
100
Systemic Hypertension In individuals carrying the p.Glu545Val variant, hypertension was considerably more prevalent in those with CKD than in those without clinical evidence of kidney disease (97% vs 30%; P , 0.001). In the unbiased comparison of individuals prospectively genotyped on family screening, the prevalence of hypertension was significantly higher in those harboring the p.Glu545Val variant than in their B
80
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80
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Survival Function Censored
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Figure 3. Kaplan-Meier estimates of actuarial (A) renal and (B) overall survival from birth for the 50 individuals identified with the FGA p.Glu545Val variant. Twenty-one patients developed end-stage renal disease and 14 died. Am J Kidney Dis. 2017;70(2):235-243
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genetically unaffected siblings (61% vs 31%; P , 0.02), but in those with high blood pressure, age at diagnosis and severity of hypertension did not differ between the 2 groups. Notably, among individuals with the p.Glu545Val variant, the prevalence of hypertension was 1.7-fold greater than the prevalence of CKD (Table 2).
DISCUSSION In this large-scale systematic study, we used 3 distinct but complementary case-finding protocols and identified the p.Glu545Val variant of AFib in 50 individuals belonging to 13 apparently unrelated families, all originating from neighboring villages in the district of Braga, northwestern Portugal. For this reason, possible confounding effects of environmental variation upon the phenotypic expression of the amyloidogenic p.Glu545Val variant are minimized in our cohort. We have previously reported that AFibE526V (p.Glu545Val) amyloidosis accounted for 3.9% (4 of 102) of the cases of amyloid nephropathy, in a consecutive series of diagnostic native kidney biopsies performed over 36 years, at Centro Hospitalar de São João.18 This percentage does not significantly differ from the 1.7% (87 of 5,100) found in a much Table 2. Demographic Characteristics and Prevalence of CKD and Hypertension by FGA Genotype Among 7 Portuguese Families With AFibE526V (p.Glu545Val) Amyloidosis p.Glu545Val Negative (n 5 35)
Male sex Age at genetic screening, y CKD Hypertension Age at diagnosis, y SBP, mm Hg DBP, mm Hg Antihypertensive therapy RAS inhibition Combination therapyb
14 (40) 42.0 6 14.3 1 (3) 11 (31) 45.7 6 12.7 150.6 6 12.0 89.4 6 8.3 10 (91) 8 (80) 5 (50)
p.Glu545Val Positive (n 5 31a)
P
14 (45) 0.7 48.9 6 17.3 0.08 11 (36) ,0.001 19 (61) 0.02 50.4 6 10.6 0.3 150.9 6 17.4 0.9 85.0 6 13.2 0.3 14 (74) 0.5 11 (85) 0.5 5 (39) 0.4
Note: Values for categorical variables are given as number (percentage); values for continuous variables, as mean 6 standard deviation. P values are from c2 test for categorical variables and t test for continuous variables. Individuals harboring the p.Glu545Val variant were compared with their corresponding negative relatives, within the same family. Abbreviations: CKD, chronic kidney disease; DBP, diastolic blood pressure; RAS, renin-angiotensin system; SBP, systolic blood pressure. a Because relatives lacking the p.Glu545Val variant were available in only 7 of 13 families with AFibE526V (p.Glu545Val) amyloidosis, not all 50 patients with the p.Glu545Val are represented in this table. b Combination therapy was defined by the use of 2 or more antihypertensive drugs. RAS inhibition included angiotensinconverting enzyme inhibitors or angiotensin II receptor blockers. 240
larger series from the National Amyloidosis Centre in the United Kingdom,25 which included tissue biopsies other than the kidney. Results of the present study show that AFibE526V (p.Glu545Val) amyloidosis is a relatively common but frequently overlooked cause of ESRD in families living or originating from a circumscribed geographic area in the northwest of Portugal. The point prevalence of ESRD due to AFibE526V (p.Glu545Val) amyloidosis could be estimated at w4.5% among the local hemodialysis population, and it was the most common amyloid nephropathy, even considering the relatively high prevalence of hereditary transthyretin amyloidosis in that region.26-28 The typical natural history of AFibE526V (p.Glu545Val) amyloidosis in our cohort was of early-onset hypertension, followed by proteinuric CKD, eventually progressing to ESRD after 5 years. The pathophysiology of hypertension in AFib amyloidosis is unclear: although it has been regarded as secondary to CKD,3 other investigators have proposed that vascular deposits of amyloid, leading to impaired endothelial function, might be the first step in the disease process.4,29 Our observation that the diagnosis of high blood pressure preceded any evidence of kidney involvement in most patients is in line with the latter hypothesis. However, the etiologic interpretation of the hypertensive disease in our patients, as well as assignment of the cause of CKD, is confounded by the high prevalence of essential hypertension in the Portuguese adult population, reaching 48.4% in people aged 35 to 64 years,30,31 and the relatively high frequency of the diagnosis of hypertensive nephrosclerosis in the prevalent Portuguese dialysis population.32 In our cohort, hypertension had a higher prevalence than in the age-matched general population, and AFibE526V (p.Glu545Val) amyloidosis was misdiagnosed as hypertensive nephrosclerosis in 3 of 12 (25%) patients on renal replacement therapy. The rate of decline in kidney function observed in our patients was slower than that reported by Gillmore et al3 in theirs, as well as in contrast to other types of amyloidosis,3 but it compares unfavorably with other more common causes of progressive CKD, such as diabetic nephropathy and hypertensive nephrosclerosis.33 During follow-up, most of the patients with AFibE526V (p.Glu545Val) amyloidosis diagnosed developed severe clinical manifestations attributable to extrarenal amyloid deposition in the heart, liver, spleen, colon, and ileum. Cardiac involvement has been documented in a patient who developed restrictive cardiomyopathy a few years after kidney transplantation.19 This is in line with genotype-phenotype correlation described for the p.Glu545Val variant4,11,34,35 and contrasts with the absence of extrarenal amyloidosis in Am J Kidney Dis. 2017;70(2):235-243
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AFibR554L (p.Arg573Leu).36 The prevalence of cerebrovascular disease was high, with 11 of the 30 (37%) patients with AFibE526V (p.Glu545Val) amyloidosis in this study reporting at least one stroke event after 55 years of age. The contribution of vascular fibrinogen amyloid deposits to the pathophysiology of cerebrovascular disease in our patients is confounded by the high incidence of stroke in northern Portugal37 and the increasing risk for stroke with CKD progression, which is substantially higher in patients with ESRD.38,39 About 39% of the 13 probands with AFibE526V (p.Glu545Val) amyloidosis were not aware of a family history of ESRD or amyloidosis. Although it has been suggested by other investigators that AFib amyloidosis has a reduced penetrance,3 this might not be the case in our population because the proper interpretation of those data is confounded by the late occurrence of the most clinically severe manifestations of the disease and the much shorter life expectancy only a few generations ago. In 1940, for example, the life expectancy in Portugal was 49.1 years for men and 53.6 years for women,40 and complications of AFib amyloidosis might not have been recognized or have been misinterpreted as hypertensive. Many of the first-degree relatives of the probands were living abroad (eg, Brazil, France, United States, United Kingdom, and Switzerland). This contributed to the low success of cascade genetic screening— which was carried out in ,50% of the at-risk individuals—and to the observation of AFibE526V (p.Glu545Val) amyloidosis in immigrant Portuguese communities, such as in France.41 AFib amyloidosis is largely a kidney disease, and outcomes after kidney transplantation are fairly good (median transplant survival, about 6 years, limited primarily by recurrent renal deposition of amyloid).3,42 Notably, our patient who was homozygous for the variant lived for 16 years with a functioning kidney transplant.19 It has been demonstrated that combined liver-kidney transplantation eliminates the amyloidogenic variant, preventing additional amyloid deposition.4,43,44 However, the high risk for early perioperative mortality with combined liver-kidney transplantation should be properly taken into account because long-term benefits are yet to be shown.45 Emerging immunotherapies that promote the clearance of amyloid deposits are potentially novel therapeutic agents for AFib amyloidosis.46-48 Our study has several limitations. First, the retrospectively collected data are susceptible to referral, selection, and indication biases, and criteria for the genetic testing of hemodialysis patients might have missed a few additional cases. Second, many of the patients and families, as well as their physicians, were not aware that they had or were at risk for a genetic Am J Kidney Dis. 2017;70(2):235-243
condition; the lack of awareness might have contributed to underascertainment of cases and delay of the diagnosis of affected individuals. Third, the requirement for molecular confirmation of the diagnosis led to exclude from the study cohort a number of possibly affected deceased individuals with CKD stage 3 or worse. In conclusion, we have characterized the clinical phenotype of AFibE526V (p.Glu545Val) amyloidosis in a large cohort of affected patients from a circumscribed region in northwest Portugal, where the disease is particularly frequent. Its natural history can be portrayed as early-onset hypertension, followed by proteinuric CKD, eventually progressing to ESRD, with increasing prevalence of extrarenal manifestations in older patients, most typically resulting from amyloid deposition in the heart, liver, and spleen. Due to the nonspecific nature of its major clinical complications, the diagnosis of AFibE526V (p.Glu545Val) amyloidosis requires a high index of suspicion and can be easily overlooked. Particularly in populations like the Portuguese, for whom hypertension is highly prevalent, the cause of CKD in patients with undiagnosed AFib amyloidosis may be erroneously attributed to hypertensive nephrosclerosis. Increasing awareness of this disease among local primary care physicians and nephrologists is critical to minimize its under-recognition, and genetic screening of all at-risk relatives of affected individuals is the most effective approach to early diagnosis. This allows the timely institution of therapies directed at minimizing the cardiovascular disease, as well as to make more appropriate decisions about the indication for solid-organ transplantation. Furthermore, with the emergence of novel disease-modifying therapies, it will be greatly desirable for patients to start treatment before irreversible tissue injury or organ failure. Finally, couples at risk should be informed about the possibility of preimplantation or prenatal diagnosis for primary prevention of AFibE526V (p.Glu545Val) amyloidosis in their progenies.
ACKNOWLEDGEMENTS This work is part of the PhD research project of Dr Tavares, supervised by Dr Lobato and co-supervised by Dr Oliveira. We thank the patients and their relatives for the invaluable collaboration in this study; NephroCare–Portugal, for having allowed the ascertainment of patients with AFib amyloidosis in the dialysis clinics of Braga and Fafe, Portugal; Hemoatlântico, for having allowed the evaluation of patients in the dialysis clinic of Vila Verde, Portugal; Dr Joaquina Coelhoso, from the Family and General Medicine, Centro de Saúde Vieira do Minho, Braga, Portugal, for collaboration in genealogic evaluation; and Dr Pedro Rodrigues Pereira, from the Department of Pathology, Centro Hospitalar de São João, Porto, Portugal, for collaboration in histologic evaluation of kidney biopsies. Support: This work was partially supported by National Funds through the FCT–Fundação para a Ciência e Tecnologia 241
Tavares et al (Portuguese national funding agency for science, research and technology) in the frameworks of the PEst-OE/SAU/UI0215/2014 project–Unit for Multidisciplinary Research in Biomedicine– UMIB/ICBAS/UP; and by a research grant from the Portuguese Society of Nephrology. The funders had no role in study design; collection, analysis, and interpretation of data; writing the report; and the decision to submit the report for publication. Financial Disclosure: The authors declare that they have no other relevant financial interests. Contributions: Research idea and study design: IT, LL; data acquisition: IT, LM, LR, JS, JP, PPC, LL; data analysis/interpretation: IT, JPO, PPC, LL; statistical analysis: JPO, AP; supervision or mentorship: LL. Each author contributed important intellectual content during manuscript drafting or revision and accepts accountability for the overall work by ensuring that questions pertaining to the accuracy or integrity of any portion of any the work are appropriately investigated and resolved. IT and LL take responsibility that this study has been reported honestly, accurately, and transparently; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned have been explained. Peer Review: Evaluated by 2 external peer reviewers, a Statistical Editor, a Co-Editor, and Editor-in-Chief Levey.
SUPPLEMENTARY MATERIAL Table S1: Comorbidities and cause of death in those with CKD and the FGA p.Glu545Val variant. Note: The supplementary material accompanying this article (http://dx.doi.org/10.1053/j.ajkd.2017.01.048) is available at www.ajkd.org
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11. Mourad G, Delabre JP, Garrigue V. Cardiac amyloidosis with the E526V mutation of the fibrinogen A alpha-chain. N Engl J Med. 2008;359(26):2847-2848. 12. Eriksson M, Schonland S, Bergner R, et al. Three German fibrinogen Aalpha-chain amyloidosis patients with the p. Glu526Val mutation. Virchows Arch. 2008;453(1):25-31. 13. Machado JR, Silva MV, Neves PD, et al. Fibrinogen A alpha-chain amyloidosis: report of the first case in Latin America. Amyloid. 2013;20(1):52-55. 14. Zhen DB, Swiecicki PL, Zeldenrust SR, Dispenzieri A, Mauermann ML, Gertz MA. Frequencies and geographic distributions of genetic mutations in transthyretin- and non-transthyretinrelated familial amyloidosis. Clin Genet. 2015;88(4):396-400. 15. Yao Y, Wang SX, Zhang YK. [Hereditary fibrinogen Aalpha-chain amyloidosis caused by the E526V mutation: a case report and literature review]. Beijing Da Xue Xue Bao. 2014;46(5): 802-804. 16. Human Genome Variation Society. Guidelines for mutation nomenclature. http://www.hgvs.org/mutnomen. Accessed January 12, 2016. 17. Sipe JD, Benson MD, Buxbaum JN, et al. Nomenclature 2014: amyloid fibril proteins and clinical classification of the amyloidosis. Amyloid. 2014;21(4):221-224. 18. Tavares I, Vaz R, Moreira L, et al. Renal amyloidosis: classification of 102 consecutive cases. Port J Nephrol Hypertens. 2014;28(3):201-209. 19. Tavares I, Lobato L, Matos C, et al. Homozygosity for the E526V mutation in fibrinogen A alpha-chain amyloidosis: the first report. Case Rep Nephrol. 2015;2015:919763. 20. Levey AS, Stevens LA, Schmid CH, et al. A new equation to estimate glomerular filtration rate. Ann Intern Med. 2009;150(9):604-612. 21. Kidney Disease: Improving Global Outcomes (KDIGO) CKD Work Group. KDIGO 2012 clinical guideline for the evaluation and management of chronic kidney disease. Kidney Int Suppl. 2013;3:S1-S150. 22. World Medical Association. Ethical principles for medical research involving human subjects. http://www.wma.net/en/3 0publications/10policies/b3/17c.pdf. Accessed December 12, 2016. 23. National Center for Biotechnology Information. Nucleotide database. https://www.ncbi.nlm.nih.gov/nuccore/NM_000508. Accessed January 12, 2016. 24. National Center for Biotechnology Information. Protein database. https://www.ncbi.nlm.nih.gov/protein/NP_000499. Accessed January 12, 2016. 25. Wechalekar AD, Gillmore JD, Hawkins PN. Systemic amyloidosis. Lancet. 2016;387(10038):2641-2654. 26. Lobato L, Beirao I, Guimaraes SM, et al. Familial amyloid polyneuropathy type I (Portuguese): distribution and characterization of renal amyloid deposits. Am J Kidney Dis. 1998;31(6):940-946. 27. Lobato L, Beirao I, Silva M, et al. Familial ATTR amyloidosis: microalbuminuria as a predictor of symptomatic disease and clinical nephropathy. Nephrol Dial Transplant. 2003;18(3):532-538. 28. Lobato L, Beirao I, Silva M, et al. End-stage renal disease and dialysis in hereditary amyloidosis TTR V30M: presentation, survival and prognostic factors. Amyloid. 2004;11(1):27-37. 29. Picken MM. Fibrinogen amyloidosis: the clot thickens! Blood. 2010;115(15):2985-2986. 30. Polonia J, Martins L, Pinto F, Nazare J. Prevalence, awareness, treatment and control of hypertension and salt intake in Portugal: changes over a decade. The PHYSA study. J Hypertens. 2014;32(6):1211-1221.
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Unrecognized Fibrinogen A a-Chain Amyloidosis 31. Macedo ME, Lima MJ, Silva AO, Alcantara P, Ramalhinho V, Carmona J. Prevalence, awareness, treatment and control of hypertension in Portugal: the PAP study. J Hypertens. 2005;23(9):1661-1666. 32. Sociedade Portuguesa de Nefrologia. Gabinete de Registo da Doença Renal Terminal. http://www.spnefro.pt/tratamento_da_ doenca_renal_terminal. Accessed March 8, 2016. 33. National Kidney Foundation. K/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification. Am J Kidney Dis. 2002;39(2)(suppl 1): S1-S266. 34. Zeldenrust S, Gertz M, Uemichi T, et al. Orthotopic liver transplantation for hereditary fibrinogen amyloidosis. Transplantation. 2003;75(4):560-561. 35. Legris T, Daniel L, Moal V. Delayed diagnosis of fibrinogen Aalpha-chain amyloidosis after dual heart-kidney transplantation. Transpl Int. 2013;26(1):e1-e3. 36. Haidinger M, Werzowa J, Kain R, et al. Hereditary amyloidosis caused by R554L fibrinogen Aalpha-chain mutation in a Spanish family and review of the literature. Amyloid. 2013;20(2):72-79. 37. Correia M, Magalhaes R, Silva MR, Matos I, Silva MC. Stroke types in rural and urban northern Portugal: incidence and 7-year survival in a community-based study. Cerebrovasc Dis Extra. 2013;3(1):137-149. 38. Chillon JM, Massy ZA, Stengel B. Neurological complications in chronic kidney disease patients. Nephrol Dial Transplant. 2016;31(10):1606-1614. 39. Tonelli M, Karumanchi SA, Thadhani R. Epidemiology and mechanisms of uremia-related cardiovascular disease. Circulation. 2016;133(5):518-536.
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40. Oliveira IT, Mendes MF. A diferença de esperança de vida entre homens e mulheres: Portugal de 1940 a 2007. Análise Social. 2010;XLV:115-138. 41. Delabre JP, Pageaux GP, Le Quellec A, Raynaud P, Grateau G, Mourad G. [A preemptive combined liver-kidney transplantation in Aalpha fibrinogen chain renal amyloidosis]. Nephrol Ther. 2009;5(2):139-143. 42. Sethi S, Fervenza FC, Miller D, Norby S, Leung N. Recurrence of amyloidosis in a kidney transplant. Am J Kidney Dis. 2010;56(2):394-398. 43. Gillmore JD, Booth DR, Rela M, et al. Curative hepatorenal transplantation in systemic amyloidosis caused by the Glu526Val fibrinogen alpha-chain variant in an English family. QJM. 2000;93(5):269-275. 44. Pinney JH, Lachmann HJ, Sattianayagam PT, et al. Renal transplantation in systemic amyloidosis-importance of amyloid fibril type and precursor protein abundance. Am J Transplant. 2013;13(2):433-441. 45. Gillmore JD, Lachmann HJ, Wechalekar A, Hawkins PN. Hereditary fibrinogen A alpha-chain amyloidosis: clinical phenotype and role of liver transplantation [letter]. Blood. 2010;115(21): 4313. author reply 4314-4315. 46. Sayed RH, Hawkins PN, Lachmann HJ. Emerging treatments for amyloidosis. Kidney Int. 2015;87(3):516-526. 47. Gillmore JD, Tennent GA, Hutchinson WL, et al. Sustained pharmacological depletion of serum amyloid P component in patients with systemic amyloidosis. Br J Haematol. 2010;148(5): 760-767. 48. Richards DB, Cookson LM, Berges AC, et al. Therapeutic clearance of amyloid by antibodies to serum amyloid P component. N Engl J Med. 2015;373(12):1106-1114.
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F
ibrinogen A a-chain (AFib) amyloidosis is a systemic disease caused by extracellular deposition of insoluble amyloid fibrils composed of abnormal fibrinogen, arising from autosomal-dominant mutations in the gene encoding AFib (FGA).1-5 Patients with AFib amyloidosis invariably develop chronic kidney disease (CKD), typically progressing to end-stage renal disease (ESRD) within 5 years of recognition of renal involvement.3 Diagnosis is based on the occurrence of proteinuric nephropathy, positive family history, identification of amyloid deposits in affected tissues From the 1Department of Nephrology, Centro Hospitalar de São João; 2Group of Research and Development in Nephrology and Infectious Diseases, Institute of Biomedical Engineering–I3S, and 3 Department of Genetics, Faculty of Medicine, University of Porto; 4 Department of Human Genetics, National Health Institute Doutor Ricardo Jorge; 5Department of Nephrology, Centro Hospitalar do Porto, Hospital de Santo António; 6Unit for Multidisciplinary Research in Biomedicine, Instituto de Ciências Biomédicas Abel Salazar, University of Porto, Porto; 7Dialysis Clinic of Braga, NephroCare–Portugal, Braga; and 8Dialysis Clinic of Fafe, NephroCare–Portugal, Fafe, Portugal. Am J Kidney Dis. 2017;70(2):235-243
by immunohistochemistry or mass spectrometry, and detection of an FGA amyloidogenic genetic variant.1-6 Incomplete penetrance may complicate the diagnosis of AFib amyloidosis and should be taken into account in genetic counseling. To date, 13 amyloidogenic FGA variants have been described,7 accounting for 8% of hereditary amyloidoses.8 Although AFib amyloidosis was originally described in 1993 in a Peruvian kindred, segregating with a mutation in FGA identified as p.Arg554Leu (ie, indicating a substitution of arginine by leucine at amino Received May 18, 2016. Accepted in revised form January 3, 2017. Originally published online March 27, 2017. Address correspondence to Isabel Tavares, MD, Department of Nephrology, Centro Hospitalar de São João, Alameda Professor Hernâni Monteiro, 4200-319 Porto, Portugal. E-mail: isabel. [email protected] 2017 by the National Kidney Foundation, Inc. 0272-6386 http://dx.doi.org/10.1053/j.ajkd.2017.01.048
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acid 554),1 the most common FGA amyloidogenic variant reported worldwide is one described as p.Glu526Val (substitution of glutamate by valine at amino acid 526), having been identified in a Canadian kindred of Polish origin9 and in families from the United Kingdom,3,10 France,11 Germany,12 Brazil,13 United States,14 and China.15 According to recommendations of the Human Genome Variation Society,16 the p.Arg554Leu and p.Glu526Val variants should be described, respectively, as p.Arg573Leu and p.Glu545Val (ie, considering the translation initiation site as amino acid 1, instead of basing the numbering on the cleaved protein). In line with this, the Nomenclature Committee of the International Society of Amyloidosis17 recommends that hereditary amyloidosis associated with the FGA p.Glu545Val variant should be designated AFibE526V (p.Glu545Val) amyloidosis, and this nomenclature will be followed hereinafter. In Portugal, the first patient with AFib amyloidosis was reported in 2004,2 and the p.Glu545Val variant was found to be the causative mutation. Subsequently, 4 other apparently unrelated patients,18 including a woman with a homozygous mutation,19 were given a diagnosis of AFib amyloidosis by retrospective immunohistochemical characterization of 102 kidney
biopsies showing amyloid nephropathy, retrieved from the archive of Centro Hospitalar de São João, a major university hospital in Porto, in the northwest of Portugal. Because all 4 patients carried the p.Glu545Val variant and were from the same confined geographic area, we hypothesized that AFibE526V (p.Glu545Val) amyloidosis may be an underdiagnosed cause of ESRD in that region and carried out targeted genetic screening among the local outpatient hemodialysis population, offering cascade genetic screening to at-risk relatives of all identified cases. We report demographic and clinical features of individuals carrying the FGA p.Glu545Val variant identified in this study, irrespective of their medical condition, and characterize the natural history and major outcomes of AFibE526V (p.Glu545Val) amyloidosis.
METHODS Study Design, Participant Ascertainment, and Data Collection Participants enrolled in this study were ascertained by 3 distinct sequential case-finding protocols (Fig 1), starting with the 4 patients who were retrospectively given diagnoses of AFibE526V (p.Glu545Val) amyloidosis, by review and immunohistochemical classification of archived kidney biopsies showing amyloid nephropathy (phase I).18
Phase I Review of the kidney biopsy registry of a major university hospital [1977-2013]: N=3,264
Histological diagnosis of amyloid nephropathy N=102
Immunohistochemical diagnosis of amyloid nephropathy due to AFib amyloid fibril protein N=4
Phase II
Phase III
Genetic diagnosis of AFib amyloidosis due to the FGA p.Glu545Val variant N=4
(all patients from a confined region in the district of Braga, NW Portugal)
Review of the prevalent patient databases of 3 local outpatient hemodialysis clinics [2005-2010]: N=267
13 unrelated families diagnosed with AFib amyloid nephropathy (2005-2014) FGA p.Glu545Val N=12 At-risk relatives N=167
Accepted genetic screening N=69
Accepted genetic screening N=122
Non-stringently defined or undetermined ESRD etiology or unclassified amyloid nephropathy N=122
Exclusion criteria: • etiology of ESRD stringently defined: N=126 • no informed consent: N=19
FGA p.Glu545Val N=34
Figure 1. Study design shows setting, eligibility, and results of included cases. Targeted genetic testing led to the identification of 50 individuals with the FGA p.Glu545Val variant in the same geographical region. Abbreviations: AFib, fibrinogen A a-chain; ESRD, end-stage renal disease; FGA, fibrinogen A a-chain gene; NW, northwest. 236
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Unrecognized Fibrinogen A a-Chain Amyloidosis The observation that the families of all 4 probands originated from neighboring villages in the district of Braga prompted a casefinding study among the local regular hemodialysis population (phase II). To this end, 267 prevalent patients with ESRD, 18 years or older, and undergoing hemodialysis in 3 outpatient clinics in the district of Braga between January 2005 and June 2010 were evaluated for eligibility for genetic screening. Criteria for offering p.Glu545Val genotyping included CKD of undetermined or nonstringently defined cause (diabetic nephropathy in the absence of diabetic retinopathy, hypertensive nephrosclerosis, and glomerular and interstitial diseases in the absence of histologic confirmation) or a diagnosis of unclassified amyloid nephropathy. Exclusion criteria were a stringently defined CKD diagnosis, either biopsy proven or inferred from a highly specific clinical context, or refusal or inability to provide informed consent. When a patient carrying the p.Glu545Val variant was identified, the relevant demographic and clinical data were collected, including age at onset of clinical manifestations; renal laboratory data, and age at the initiation of renal replacement therapy; history of vascular, gastrointestinal, and neurologic morbidity and their respective clinical outcomes; and age at death. Additional clinical data were retrospectively collected by review of archived medical records, the oldest available dating as far back as 1973, or by interviewing the patient’s relatives. A detailed genogram capturing at least 3 generations was drawn for every patient diagnosed in phases I and II to check for possible relatedness among their families, as well as to identify genetically at-risk individuals. The latter were offered genetic counseling and those who eventually accepted genetic testing (phase III) underwent comprehensive baseline clinical assessment, including repeat blood pressure readings, plasma creatinine and proteinuria measurements, and kidney ultrasonography. Follow-up specialized medical care was offered to all participants in this study, and the relevant clinical events in their natural histories were prospectively collected until December 2014. For the purpose of this study, the diagnosis of AFibE526V (p.Glu545Val) nephropathic amyloidosis did not require histologic confirmation of amyloid deposition on a kidney biopsy; patients harboring the p.Glu545Val variant were regarded as affected if they also had CKD, as defined in the next paragraph, whereas those who did not display significant manifestations of renal involvement were designated as genetic carriers. Furthermore, FGA genotyping was a requisite for enrollment in this study of the genetically at-risk individuals identified by pedigree analysis, irrespective of medical history or current clinical condition. Criteria for diagnosis of CKD were proteinuria with protein excretion $ 0.15 g/24 h or estimated glomerular filtration rate (eGFR) , 60 mL/min/1.73 m2 for 3 or more months, as calculated by the CKD-EPI (CKD Epidemiology Collaboration) creatinine equation.20 Staging of CKD was according to the KDIGO (Kidney Disease: Improving Global Outcomes) guideline.21 Systemic hypertension was defined as systolic blood pressure $ 140 mm Hg and/or diastolic blood pressure $ 90 mm Hg or current antihypertensive drug treatment. The clinical diagnosis of hypertension was based on a blood pressure assessment made at the time of blood sampling for FGA genotyping, as well as on historical records and/or prospective measurements obtained within 1 year of the baseline evaluation. In the absence of CKD, hypertensive patients carrying the p.Glu545Val variant were not given a diagnosis of nephropathic amyloidosis but instead were classified as genetic carriers. This study was conducted in accordance with the Ethical Principles for Medical Research Involving Human Subjects adopted by the World Medical Association,22 and its design and research protocol were reviewed and approved by the Centro Hospitalar de São João Health Ethics Commission (resolution no. 90/2012). Written informed consent was obtained from all participants. Am J Kidney Dis. 2017;70(2):235-243
Molecular Analysis Genomic DNA was extracted from peripheral-blood samples with the Prepito DNA Blood250 Kit (Perkin Elmer) according to the manufacturer’s instructions. The p.Glu545Val variant, coded for by c.1634A.T (an adenine to thymidine substitution at nucleotide 1,634 of the coding sequence, in exon 5 of the FGA gene) was screened by polymerase chain reaction amplification followed by Sanger sequencing using forward primer 50 -CTTCATCTGCAGTCAGCGATC-30 and reverse primer 50 -GAGATTTAGCATGGCCTCTC-30 . Polymerase chain reaction amplification was carried out in a Veriti thermal cycler (Applied Biosystems) with the following conditions: 95 C for 5 minutes, 35 amplification cycles (of denaturation at 95 C for 1 minute, annealing at 61 C for 1 minute, and extension at 72 C for 1 minute), and terminal extension at 72 C for 10 minutes. Polymerase chain reaction products were analyzed with the QIAXcel system (QIAGEN), purified with Agencourt AMPure XP–PCR Purification (Beckman Coulter), and sequenced on an automated ABI PRISM TM 3500 Genetic Analyser (Applied Biosystems) using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The National Center for Biotechnology Information reference sequences NM_00050823 and NP_00049924 were used as the references for the FGA complementary DNA and protein sequences, respectively.
Statistical Analyses Categorical variables were expressed as number and percentage; continuous variables were expressed as mean with standard deviation or as median with interquartile range, depending on whether they were normally distributed. Individuals carrying the p.Glu545Val variant were grouped according to the case-finding protocol in which they were identified, and the 3 cohorts were compared for relevant demographic and clinical data using Fisher exact test for categorical variables and 1-way analysis of variance or Kruskal-Wallis test for continuous variables, depending on goodness of fit to normal distribution. Prevalence, age of onset, and severity of hypertension were compared in p.Glu545Val carrier and noncarrier siblings, prospectively identified in family screening. To minimize ascertainment bias, family probands (n 5 10) were excluded from these analyses. The t test or c2 test, respectively, was used for comparing normally distributed continuous variables or categorical variables. The relationship between proteinuria and eGFR in the 30 patients with CKD was analyzed by Pearson correlation coefficient, using the first available proteinuria value with protein excretion $ 0.15 g/24 h. Decline in eGFR over time was analyzed by mixed-effects model in the 20 patients with CKD who had at least 2 serum creatinine measurements obtained more than 1 year apart. Absolute risks for major adverse outcomes (ie, ESRD and death) were estimated for the entire cohort (n 5 50), using Kaplan-Meier curves to determine renal and overall mean survival, censoring for death in the case of ESRD. P , 0.05 was considered statistically significant. All statistical analyses were performed using the IBM SPSS, version 20.0, software package for Macintosh.
RESULTS Study Population As shown in Fig 1, a total of 195 individuals eventually underwent genetic testing for the FGA p.Glu545Val variant, including the 4 patients retrospectively given diagnoses of AFib amyloidosis in phase I; all the eligible 122 hemodialysis patients enrolled in phase II; and 69 of 167 (41.3%) first-degree relatives of patients with AFibE526V (p.Glu545Val) amyloidosis who were identified in the 2 case-finding 237
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protocols. All those individuals were of Portuguese ancestry, with family roots in the district of Braga. The diagnosis of AFibE526V (p.Glu545Val) amyloidosis was established by molecular analysis in all 4 patients enrolled in phase I and in 12 of 122 (9.8%) hemodialysis patients screened in phase II. Of the latter patients, 6 were given diagnoses of unclassified amyloid nephropathy, whereas 3 had received a diagnosis of hypertensive nephrosclerosis and the remaining 3 had been classified as having ESRD of undetermined cause. On family screening, 34 of 69 (49%) individuals were found to be heterozygous for the p.Glu545Val variant, including 14 (41%) patients with evidence of CKD, none of whom was unaware of their clinical condition. Overall, the p.Glu545Val variant was identified in 50 individuals from 13 apparently unrelated families. Notably, 7 individuals from 3 unrelated families had died while receiving regular hemodialysis treatment before the beginning of this study and therefore did not have a definite genetic diagnosis. Characteristics of Individuals Carrying the FGA p.Glu545Val Variant
summarized for the overall cohort and for participants grouped according to the case-finding protocol in which they were identified. Mean age at genetic testing was 52.0 6 16.8 years. Hypertension was detected in 35 (70%) individuals at a mean age of 51.0 6 10.4 years and was present even in the absence of kidney disease. Thirty (60%) individuals developed CKD at a mean age of 56.7 6 12.0 years, presenting with proteinuria with protein excretion . 3 g/24 h and eGFR , 60 mL/min/1.73 m2 in more than half the cases, but only 17 (57%) such patients had undergone a diagnostic kidney biopsy. In phase III, a total of 20 of 34 (59%) individuals with the p.Glu545Val variant had no evidence of CKD at a mean age of 38.4 6 9.7 years and were therefore classified as genetic carriers. These individuals were significantly younger than their 14 relatives with CKD diagnosed, with a mean age difference of 18.4 years. Five (25%) individuals with no evidence of CKD had high blood pressure diagnosed as essential hypertension. Kidney Disease and Other Major Clinical Outcomes
The relevant demographic and clinical characteristics of individuals with the p.Glu545Val variant diagnosed in this study, irrespective of clinical condition, are presented in Table 1. The data are
All individuals with CKD presented with proteinuria. As shown in Fig 2, the severity of proteinuria associated inversely with eGFR (r 5 20.426; P 5 0.02), and mean change in eGFR was 24.39
Table 1. Diagnostic Assessment and Main Clinical Characteristics of Individuals With the FGA p.Glu545Val Variant Ascertainment Overall (N 5 50)
Kidney Biopsy Registry (n 5 4)a
Hemodialysis Screening (n 5 12)a
Family Screening (n 5 34)
P
Male sex Age at genetic testing, y Hypertension Age at diagnosis, y
22 (44) 52.0 6 16.8 35 (70) 51.0 6 10.4
2 (50) 57.5 6 16.6 4 (100) 52.8 6 12.5
6 (50) 67.2 6 12.2 12 (100) 52.6 6 11.8
14 (41) 46.0 6 14.7 19 (56) 49.7 6 9.5
0.8 ,0.001 0.006 0.7
Kidney disease presentation Age at presentation, y Proteinuria ,1 g/24 h 1-3 g/24 h .3 g/24 h eGFR ,60 mL/min/1.73 m2 $60 mL/min/1.73 m2
30 (60) 56.7 6 12.0
4 (100) 53.0 6 12.5
12 (100) 59.7 6 10.9
14 (41) 55.2 6 13.1
,0.001 0.5 0.05
9 (30) 4 (13) 17 (57)
0 (0) 1 (25) 3 (75)
1 (8) 2 (17) 9 (75)
8 (57) 1 (7) 5 (36)
19 (63) 11 (37)
3 (75) 1 (25)
12 (100) 0 (0)
4 (29) 10 (71)
Renal replacement therapy Age at initiation, y Dialysis vintage, mob Kidney transplantation
21 (42) 61.4 6 11.3 48.0 [27.0-90.5] 2 (10)
4 (100) 55.0 6 12.6 13.0 [3.8-203.0] 1 (25)
12 (100) 64.8 6 11.2 77.5 [47.2-91.2] 1 (8)
5 (15) 58.2 6 9.0 35.0 [18.5-67.0] 0 (0)
0.02
,0.001 0.3 0.1 NAc
Note: Values for categorical variables are given as number (percentage); values for continuous variables, as mean 6 standard deviation if normally distributed or median [interquartile range] if non-normally distributed. P values are from Fisher exact test for categorical variables and 1-way analysis of variance or Kruskal-Wallis for continuous variables, as appropriate. Abbreviations: eGFR, estimated glomerular filtration rate; NA, not applicable. a Patients ascertained by kidney biopsy registry and hemodialysis screening belonged to 13 unrelated families. b End of follow-up December 2014. c No applicability in this category. 238
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Figure 2. (A) Scatterplot of proteinuria levels by estimated glomerular filtration rate (eGFR) for the 30 individuals with chronic kidney disease ascertained through a kidney biopsy registry (n 5 4), hemodialysis patient screening (n 5 12), and cascade family screening (n 5 14), analyzed by Pearson correlation coefficient (r 5 20.426; P 5 0.02). (B) eGFR regression slope for 20 of the 30 patients with chronic kidney disease who had at least 2 serum creatinine measurements available for review.
n 5 1; and spleen, n 5 1). Fourteen patients have died, at a mean age of 70.8 6 9.6 years, and their reported immediate causes of death are also summarized in Table S1. For the 50 patients with the p.Glu545Val variant (Fig 3), estimated mean renal survival was 65.4 (95% CI, 61.8-69.0) years, and estimated mean lifetime survival was 73.7 (95% CI, 70.5-76.8) years.
(95% confidence interval [CI], 23.65 to 25.14) mL/ min/1.73 m2 per year. Ten men and 11 women progressed to ESRD, having initiated renal replacement therapy at 61.4 6 11.3 years (Table 1); 2 women underwent kidney transplantation 6 years after the onset of hemodialysis therapy. Family history of kidney disease or amyloidosis was present in 61% of probands. Table S1 (provided as online supplementary material) summarizes the most relevant comorbid conditions and severe medical events that have occurred in the 30 patients with CKD. Thirteen strokes were reported (6 events in 6 women and 7 events in 5 men), with the middle cerebral artery territory being the most commonly affected location. Mean age for first stroke was 66.4 6 8.3 years. Amyloid deposits were also identified in surgical samples from hemodialysis patients (abdominal fat, n 5 1; colon, n 5 1; ileum, A
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Systemic Hypertension In individuals carrying the p.Glu545Val variant, hypertension was considerably more prevalent in those with CKD than in those without clinical evidence of kidney disease (97% vs 30%; P , 0.001). In the unbiased comparison of individuals prospectively genotyped on family screening, the prevalence of hypertension was significantly higher in those harboring the p.Glu545Val variant than in their B
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Figure 3. Kaplan-Meier estimates of actuarial (A) renal and (B) overall survival from birth for the 50 individuals identified with the FGA p.Glu545Val variant. Twenty-one patients developed end-stage renal disease and 14 died. Am J Kidney Dis. 2017;70(2):235-243
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genetically unaffected siblings (61% vs 31%; P , 0.02), but in those with high blood pressure, age at diagnosis and severity of hypertension did not differ between the 2 groups. Notably, among individuals with the p.Glu545Val variant, the prevalence of hypertension was 1.7-fold greater than the prevalence of CKD (Table 2).
DISCUSSION In this large-scale systematic study, we used 3 distinct but complementary case-finding protocols and identified the p.Glu545Val variant of AFib in 50 individuals belonging to 13 apparently unrelated families, all originating from neighboring villages in the district of Braga, northwestern Portugal. For this reason, possible confounding effects of environmental variation upon the phenotypic expression of the amyloidogenic p.Glu545Val variant are minimized in our cohort. We have previously reported that AFibE526V (p.Glu545Val) amyloidosis accounted for 3.9% (4 of 102) of the cases of amyloid nephropathy, in a consecutive series of diagnostic native kidney biopsies performed over 36 years, at Centro Hospitalar de São João.18 This percentage does not significantly differ from the 1.7% (87 of 5,100) found in a much Table 2. Demographic Characteristics and Prevalence of CKD and Hypertension by FGA Genotype Among 7 Portuguese Families With AFibE526V (p.Glu545Val) Amyloidosis p.Glu545Val Negative (n 5 35)
Male sex Age at genetic screening, y CKD Hypertension Age at diagnosis, y SBP, mm Hg DBP, mm Hg Antihypertensive therapy RAS inhibition Combination therapyb
14 (40) 42.0 6 14.3 1 (3) 11 (31) 45.7 6 12.7 150.6 6 12.0 89.4 6 8.3 10 (91) 8 (80) 5 (50)
p.Glu545Val Positive (n 5 31a)
P
14 (45) 0.7 48.9 6 17.3 0.08 11 (36) ,0.001 19 (61) 0.02 50.4 6 10.6 0.3 150.9 6 17.4 0.9 85.0 6 13.2 0.3 14 (74) 0.5 11 (85) 0.5 5 (39) 0.4
Note: Values for categorical variables are given as number (percentage); values for continuous variables, as mean 6 standard deviation. P values are from c2 test for categorical variables and t test for continuous variables. Individuals harboring the p.Glu545Val variant were compared with their corresponding negative relatives, within the same family. Abbreviations: CKD, chronic kidney disease; DBP, diastolic blood pressure; RAS, renin-angiotensin system; SBP, systolic blood pressure. a Because relatives lacking the p.Glu545Val variant were available in only 7 of 13 families with AFibE526V (p.Glu545Val) amyloidosis, not all 50 patients with the p.Glu545Val are represented in this table. b Combination therapy was defined by the use of 2 or more antihypertensive drugs. RAS inhibition included angiotensinconverting enzyme inhibitors or angiotensin II receptor blockers. 240
larger series from the National Amyloidosis Centre in the United Kingdom,25 which included tissue biopsies other than the kidney. Results of the present study show that AFibE526V (p.Glu545Val) amyloidosis is a relatively common but frequently overlooked cause of ESRD in families living or originating from a circumscribed geographic area in the northwest of Portugal. The point prevalence of ESRD due to AFibE526V (p.Glu545Val) amyloidosis could be estimated at w4.5% among the local hemodialysis population, and it was the most common amyloid nephropathy, even considering the relatively high prevalence of hereditary transthyretin amyloidosis in that region.26-28 The typical natural history of AFibE526V (p.Glu545Val) amyloidosis in our cohort was of early-onset hypertension, followed by proteinuric CKD, eventually progressing to ESRD after 5 years. The pathophysiology of hypertension in AFib amyloidosis is unclear: although it has been regarded as secondary to CKD,3 other investigators have proposed that vascular deposits of amyloid, leading to impaired endothelial function, might be the first step in the disease process.4,29 Our observation that the diagnosis of high blood pressure preceded any evidence of kidney involvement in most patients is in line with the latter hypothesis. However, the etiologic interpretation of the hypertensive disease in our patients, as well as assignment of the cause of CKD, is confounded by the high prevalence of essential hypertension in the Portuguese adult population, reaching 48.4% in people aged 35 to 64 years,30,31 and the relatively high frequency of the diagnosis of hypertensive nephrosclerosis in the prevalent Portuguese dialysis population.32 In our cohort, hypertension had a higher prevalence than in the age-matched general population, and AFibE526V (p.Glu545Val) amyloidosis was misdiagnosed as hypertensive nephrosclerosis in 3 of 12 (25%) patients on renal replacement therapy. The rate of decline in kidney function observed in our patients was slower than that reported by Gillmore et al3 in theirs, as well as in contrast to other types of amyloidosis,3 but it compares unfavorably with other more common causes of progressive CKD, such as diabetic nephropathy and hypertensive nephrosclerosis.33 During follow-up, most of the patients with AFibE526V (p.Glu545Val) amyloidosis diagnosed developed severe clinical manifestations attributable to extrarenal amyloid deposition in the heart, liver, spleen, colon, and ileum. Cardiac involvement has been documented in a patient who developed restrictive cardiomyopathy a few years after kidney transplantation.19 This is in line with genotype-phenotype correlation described for the p.Glu545Val variant4,11,34,35 and contrasts with the absence of extrarenal amyloidosis in Am J Kidney Dis. 2017;70(2):235-243
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AFibR554L (p.Arg573Leu).36 The prevalence of cerebrovascular disease was high, with 11 of the 30 (37%) patients with AFibE526V (p.Glu545Val) amyloidosis in this study reporting at least one stroke event after 55 years of age. The contribution of vascular fibrinogen amyloid deposits to the pathophysiology of cerebrovascular disease in our patients is confounded by the high incidence of stroke in northern Portugal37 and the increasing risk for stroke with CKD progression, which is substantially higher in patients with ESRD.38,39 About 39% of the 13 probands with AFibE526V (p.Glu545Val) amyloidosis were not aware of a family history of ESRD or amyloidosis. Although it has been suggested by other investigators that AFib amyloidosis has a reduced penetrance,3 this might not be the case in our population because the proper interpretation of those data is confounded by the late occurrence of the most clinically severe manifestations of the disease and the much shorter life expectancy only a few generations ago. In 1940, for example, the life expectancy in Portugal was 49.1 years for men and 53.6 years for women,40 and complications of AFib amyloidosis might not have been recognized or have been misinterpreted as hypertensive. Many of the first-degree relatives of the probands were living abroad (eg, Brazil, France, United States, United Kingdom, and Switzerland). This contributed to the low success of cascade genetic screening— which was carried out in ,50% of the at-risk individuals—and to the observation of AFibE526V (p.Glu545Val) amyloidosis in immigrant Portuguese communities, such as in France.41 AFib amyloidosis is largely a kidney disease, and outcomes after kidney transplantation are fairly good (median transplant survival, about 6 years, limited primarily by recurrent renal deposition of amyloid).3,42 Notably, our patient who was homozygous for the variant lived for 16 years with a functioning kidney transplant.19 It has been demonstrated that combined liver-kidney transplantation eliminates the amyloidogenic variant, preventing additional amyloid deposition.4,43,44 However, the high risk for early perioperative mortality with combined liver-kidney transplantation should be properly taken into account because long-term benefits are yet to be shown.45 Emerging immunotherapies that promote the clearance of amyloid deposits are potentially novel therapeutic agents for AFib amyloidosis.46-48 Our study has several limitations. First, the retrospectively collected data are susceptible to referral, selection, and indication biases, and criteria for the genetic testing of hemodialysis patients might have missed a few additional cases. Second, many of the patients and families, as well as their physicians, were not aware that they had or were at risk for a genetic Am J Kidney Dis. 2017;70(2):235-243
condition; the lack of awareness might have contributed to underascertainment of cases and delay of the diagnosis of affected individuals. Third, the requirement for molecular confirmation of the diagnosis led to exclude from the study cohort a number of possibly affected deceased individuals with CKD stage 3 or worse. In conclusion, we have characterized the clinical phenotype of AFibE526V (p.Glu545Val) amyloidosis in a large cohort of affected patients from a circumscribed region in northwest Portugal, where the disease is particularly frequent. Its natural history can be portrayed as early-onset hypertension, followed by proteinuric CKD, eventually progressing to ESRD, with increasing prevalence of extrarenal manifestations in older patients, most typically resulting from amyloid deposition in the heart, liver, and spleen. Due to the nonspecific nature of its major clinical complications, the diagnosis of AFibE526V (p.Glu545Val) amyloidosis requires a high index of suspicion and can be easily overlooked. Particularly in populations like the Portuguese, for whom hypertension is highly prevalent, the cause of CKD in patients with undiagnosed AFib amyloidosis may be erroneously attributed to hypertensive nephrosclerosis. Increasing awareness of this disease among local primary care physicians and nephrologists is critical to minimize its under-recognition, and genetic screening of all at-risk relatives of affected individuals is the most effective approach to early diagnosis. This allows the timely institution of therapies directed at minimizing the cardiovascular disease, as well as to make more appropriate decisions about the indication for solid-organ transplantation. Furthermore, with the emergence of novel disease-modifying therapies, it will be greatly desirable for patients to start treatment before irreversible tissue injury or organ failure. Finally, couples at risk should be informed about the possibility of preimplantation or prenatal diagnosis for primary prevention of AFibE526V (p.Glu545Val) amyloidosis in their progenies.
ACKNOWLEDGEMENTS This work is part of the PhD research project of Dr Tavares, supervised by Dr Lobato and co-supervised by Dr Oliveira. We thank the patients and their relatives for the invaluable collaboration in this study; NephroCare–Portugal, for having allowed the ascertainment of patients with AFib amyloidosis in the dialysis clinics of Braga and Fafe, Portugal; Hemoatlântico, for having allowed the evaluation of patients in the dialysis clinic of Vila Verde, Portugal; Dr Joaquina Coelhoso, from the Family and General Medicine, Centro de Saúde Vieira do Minho, Braga, Portugal, for collaboration in genealogic evaluation; and Dr Pedro Rodrigues Pereira, from the Department of Pathology, Centro Hospitalar de São João, Porto, Portugal, for collaboration in histologic evaluation of kidney biopsies. Support: This work was partially supported by National Funds through the FCT–Fundação para a Ciência e Tecnologia 241
Tavares et al (Portuguese national funding agency for science, research and technology) in the frameworks of the PEst-OE/SAU/UI0215/2014 project–Unit for Multidisciplinary Research in Biomedicine– UMIB/ICBAS/UP; and by a research grant from the Portuguese Society of Nephrology. The funders had no role in study design; collection, analysis, and interpretation of data; writing the report; and the decision to submit the report for publication. Financial Disclosure: The authors declare that they have no other relevant financial interests. Contributions: Research idea and study design: IT, LL; data acquisition: IT, LM, LR, JS, JP, PPC, LL; data analysis/interpretation: IT, JPO, PPC, LL; statistical analysis: JPO, AP; supervision or mentorship: LL. Each author contributed important intellectual content during manuscript drafting or revision and accepts accountability for the overall work by ensuring that questions pertaining to the accuracy or integrity of any portion of any the work are appropriately investigated and resolved. IT and LL take responsibility that this study has been reported honestly, accurately, and transparently; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned have been explained. Peer Review: Evaluated by 2 external peer reviewers, a Statistical Editor, a Co-Editor, and Editor-in-Chief Levey.
SUPPLEMENTARY MATERIAL Table S1: Comorbidities and cause of death in those with CKD and the FGA p.Glu545Val variant. Note: The supplementary material accompanying this article (http://dx.doi.org/10.1053/j.ajkd.2017.01.048) is available at www.ajkd.org
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