Association of the Transthyretin Variant V122I With Polyneuropathy Among Individuals of African Descent

BACKGROUND Hereditary transthyretin-mediated (hATTR) amyloidosis is a progressively debilitating disease caused by transthyretin (TTR) gene mutations. The V122I variant, a common pathogenic TTR mutation found primarily in individuals of West African descent, is frequently associated with cardiomyopathy. METHODS The association between the V122I variant and ICD10 diagnosis codes was assessed in the UK Biobank black subpopulation (N=6062); whether significant associations could be replicated in the Penn Medicine Biobank (N=5737) and Million Veteran Program (N=82,382) was then investigated. Cumulative incidence of common hATTR amyloidosis manifestations (polyneuropathy, carpal tunnel syndrome, cardiomyopathy, and heart failure) was estimated by V122I genotype in the UK Biobank using Cox regression controlling for age, sex, and genetic ancestry. RESULTS Phenome-wide analysis identified a significant association between V122I genotype and polyneuropathy diagnosis (odds ratio [OR]=6.4, 95% confidence interval [CI]=2.6-15.6, P=4.2x10-5) in the UK Biobank. A significant association was also observed in the Penn Medicine Biobank (OR=1.6, 95% CI=1.2-2.4, P=6.0x10-3) and the Million Veteran Program (OR=1.5, 95% CI=1.2-1.8, P=1.8x10-4). Prevalence of a polyneuropathy diagnosis among V122I carriers was 2.1%, 9.0%, and 4.8% in the UK Biobank, Penn Medicine Biobank, and Million Veteran Program, respectively. In the UK Biobank, common hATTR amyloidosis manifestations were significantly enriched in V122I carriers compared with non-carriers (HR=2.8; 95% CI=1.7-4.5; P=2.6x10-5). By age 75, 37.4% of V122I carriers had a diagnosis of any one of the common manifestations, including polyneuropathy (7.9%). CONCLUSIONS V122I carriers, historically associated with a predominantly cardiac phenotype of hATTR amyloidosis, have a significantly increased risk of polyneuropathy.


Introduction
Hereditary transthyretin-mediated (hATTR) amyloidosis, also known as ATTRv amyloidosis, is an underdiagnosed, progressively debilitating, and fatal disease caused by mutations in the transthyretin (TTR) gene. These pathogenic variants result in misfolding of TTR proteins which accumulate as amyloid deposits in multiple tissues throughout the body, including the heart, nerves, and gastrointestinal tract. [1][2][3] Organ involvement and symptoms of hATTR amyloidosis can vary by patient and mutation, but carriers of pathogenic variants have an increased risk of developing life-impacting polyneuropathy, cardiomyopathy, and other symptoms (eg, carpal tunnel syndrome [CTS]). 4,5 One of the most common pathogenic TTR variants is the valine-to-isoleucine substitution at position 122 (V122I; p.V142I; rs76992529), which is predominantly found in individuals of West African descent, 6 with 3%-4% of this population carrying the mutation. 7 The variant was originally identified in cardiac TTR amyloid deposits, 8 and subsequent studies demonstrated it as a common cause of heart failure (HF) among elderly African patients. 9,10 V122I hATTR amyloidosis is substantially underdiagnosed, 5 and patients are often at an advanced disease stage when finally diagnosed. This long journey to diagnosis also results in poor outcomes for these patients, with median time from diagnosis to death being 2.6 years. 9 Thus, a better understanding of early hATTR amyloidosis disease manifestations is critical to expedite diagnosis and initiate treatment. 11 Unlike some other common TTR mutations, the V122I variant has historically been associated with a predominantly cardiac phenotype.
However, a better understanding of the full range of disease manifestations associated with All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.10.20219675 doi: medRxiv preprint this variant is critical to improve disease recognition and allow maximal benefit from currently available therapies.
The present study assessed the association of the V122I genotype with ICD10 (International Statistical Classification of Diseases and Related Health Problems, 10th Revision) disease diagnoses in the UK Biobank, a large population-based prospective cohort study. 12 The UK Biobank study does not recruit participants based on any disease outcome, thus affording the unique opportunity to analyze the V122I variant in a population not influenced by referral bias, allowing better understanding of early disease manifestations and variant penetrance.

Significant associations were assessed for replication in the Penn Medicine Biobank and Million
Veteran Program.

Study Populations
The UK Biobank recruited ~500 000 participants in England, Wales, and Scotland between 2006 and 2010. 13 The Penn Medicine Biobank includes over 60 000 participants over age 18

V122I Genotyping
The V122I genotype was directly typed by the UK Biobank on either the UK BiLEVE or the UK Biobank Axiom arrays 15 (info score = 1). Additional genotyping details are provided in the Supplementary eAppendix. Patients with and without the V122I genotype are referred to as "carriers" and "non-carriers," respectively. The presence/absence of other TTR genotypes was not examined in these populations.

Statistical Analysis
Descriptive statistics are presented as means (standard deviations) for continuous variables and percentages for categoric variables. Continuous and categoric variables were compared between V122I carriers and non-carriers using a t-test and a Pearson's chi-squared test, respectively.

Phenome-Wide Association Analysis and Replication
To identify diagnoses associated with the V122I variant, a phenome-wide association analysis (PHEWAS) was performed on any primary or secondary inpatient ICD10 diagnosis code observed in at least 15 Black participants from the UK Biobank (n = 427 ICD10 codes). PHEWAS was performed in PLINK 16 using logistic regression controlling for age, sex, and genetic ancestry All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.10.20219675 doi: medRxiv preprint via 10 principal components. A Bonferroni-corrected P = 1.210 -4 was considered statistically significant. Significant associations with ICD9 and/or ICD10 diagnoses were assessed for replication in the Penn Medicine Biobank and Million Veteran Program using logistic regression controlling for age, sex, and genetic ancestry. Additional methodologic details are provided in the Supplementary eAppendix.

Assessment of Common hATTR Amyloidosis Manifestations in V122I Carriers
A variable was created to indicate if UK Biobank participants had been diagnosed with at least 1 of 4 common hATTR amyloidosis manifestations, independent of whether the participants had a diagnosis of hATTR amyloidosis, using the following ICD10 codes: polyneuropathy ("G62"), CTS ("G560"), cardiomyopathy ("I42"), and HF ("I50" or "I098"), hereafter termed "common hATTR amyloidosis manifestations." The association between the V122I genotype and a diagnosis of a common hATTR amyloidosis manifestation was tested using logistic regression and Cox proportional hazards regression controlling for age, sex, and genetic ancestry; P < .05 was considered statistically significant. Kaplan-Meier curves were used to estimate cumulative incidence of common hATTR amyloidosis manifestations by V122I genotype (additional methodologic details in the Supplementary eAppendix).

Characteristics of V122I Carriers With Common hATTR Amyloidosis Manifestations
V122I carriers with at least 1 common hATTR amyloidosis manifestation were compared with V122I carriers without manifestations using logistic regression, controlling for baseline age, sex, body mass index, cigarette smoking status, and genetic principal components. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The population risk of common hATTR amyloidosis manifestations attributable to the V122I genotype was calculated by multiplying the fraction of all diagnoses in V122I carriers by the risk difference between carriers and non-carriers. Risk was estimated using the cumulative incidence of the manifestations at age 75 for V122I carriers and non-carriers (additional methodologic details are described in eAppendix1 in the Supplement). Among the 243 V122I carriers, 0.8% (n = 2) were formally diagnosed with amyloidosis (ICD10 diagnosis "E85") compared with 0.1% (n = 5) of non-carriers. Both diagnosed patients were male heterozygous carriers, diagnosed at ages 75.9 and 71.9 years, respectively, and with cardiac manifestations of disease diagnosed during study follow-up. One of the 2 subjects died All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

Phenome-Wide Association Analysis
In a PHEWAS of the V122I variant in the unrelated Black UK Biobank subpopulation that included 427 ICD10 diagnosis codes, 1 significant association was identified, namely, the Logistic regression demonstrated that the V122I genotype was significantly associated with having at least 1 common hATTR amyloidosis manifestation of either polyneuropathy, CTS, cardiomyopathy, or HF (OR, 2.66, P = 1.5×10 -6 ). In total, 11.1% of V122I carriers had at least 1 common hATTR amyloidosis manifestation compared with 4.9% of non-carriers. Prevalence of All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.10.20219675 doi: medRxiv preprint each of the 4 manifestations was higher in V122I carriers than non-carriers (eFigure 3 in the Supplement) and 2 of the 3 participants homozygous for V122I were diagnosed with at least 1 of the common hATTR amyloidosis manifestations (eFigure 4 in the Supplement). Significant associations between the V122I genotype and common hATTR amyloidosis manifestations were also observed in the Million Veteran Program (eTable 2 in the Supplement).
Cardiomyopathy and HF tended to be more prevalent in V122I carriers than non-carriers at older ages ( Figures 3C and 3D), whereas polyneuropathy was more prevalent at younger ages ( Figure 3A). All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

Characteristics of V122I Carriers With Common hATTR Amyloidosis Manifestations in the UK Biobank
Multiple logistic regression demonstrated that V122I carriers with at least 1 common hATTR amyloidosis manifestation (n = 27) were more likely to be older and current/former cigarette smokers than those without common manifestations (n = 216). No sex difference was observed between those with and without any of the 4 common hATTR amyloidosis manifestations (P = .18) (eTable 4 in the Supplement).

Population Attributable Risk of Common hATTR Amyloidosis Manifestations to the V122I Variant
In the UK Biobank Black population, 16.7% of the risk of polyneuropathy diagnosis, 4.1% of the risk of a CTS diagnosis, 2.4% of the risk of a cardiomyopathy diagnosis, and 6.5% of the risk of a HF diagnosis was attributable to the V122I variant (Table 2).

Discussion
In this analysis of 3 large biobanks (UK Biobank, Penn Medicine Biobank, and the Million Veteran Program) including 2739 carriers, the V122I variant was significantly associated with a polyneuropathy diagnosis. This suggests that individuals with the V122I genotype, who were historically assumed to be predominantly at risk for cardiomyopathy, have a significantly increased risk of polyneuropathy. In the UK Biobank, by age 75, 7.9% of V122I carriers had a clinical diagnosis of polyneuropathy. Moreover, V122I carriers represent 1 in 5 of all All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.10.20219675 doi: medRxiv preprint polyneuropathy diagnoses in this subpopulation of the UK Biobank. The V122I genotype has been linked to polyneuropathy in case reports and smaller studies [17][18][19] as well as highlighted within a recent scientific statement from the American Heart Association. 20 However, to our knowledge, this is the first study to show a significant association between the V122I genotype and polyneuropathy. Additionally, an overall enrichment of common hATTR amyloidosis manifestations (polyneuropathy, CTS, cardiomyopathy, and HF) was found among V122I carriers. Despite this clear enrichment, only 2 out of 243 patients were diagnosed with amyloidosis (ICD10 "E85"), suggesting a considerable underdiagnosis of the disease that is consistent with previous literature. 4,5 Notably, the 2 participants with confirmed amyloidosis were diagnosed in their 70s, with 1 dying from cardiac amyloid less than 2 years after diagnosis.
This highlights the need for a better understanding of the early and multisystem manifestations of hATTR amyloidosis, particularly for V122I carriers who have higher mortality rates and worse prognosis than patients with other cardiac pathologies. 9,[21][22][23] To this end, the cumulative incidence of the 4 common hATTR amyloidosis manifestations was analyzed separately, finding V122I carriers had a significantly higher cumulative incidence of polyneuropathy, CTS, and HF than non-carriers. Moreover, V122I carriers were more likely than non-carriers to have polyneuropathy at a younger age and cardiac manifestations at older ages. This finding implies that diagnosing polyneuropathy may be key to earlier disease identification.
In order to identify the characteristics of participants with common hATTR amyloidosis manifestations, differences between V122I carriers with and without any diagnoses of the All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.10.20219675 doi: medRxiv preprint common hATTR amyloidosis manifestations were analyzed. V122I carriers with these diagnoses were older and more likely to be current/former smokers. However, a difference between men and women in prevalence of common hATTR amyloidosis manifestations was not found, despite previous studies identifying gender as a significant modifier of disease penetrance. 24 While the 2 amyloidosis diagnoses were both in males, diagnoses of common hATTR amyloidosis manifestations were present in equal proportions of males and females.
The results are particularly important given recent advances in therapies for hATTR amyloidosis. Two major strategies have been employed: 1) reduce serum TTR levels by inhibiting hepatic synthesis of TTR proteins through RNA interference therapeutics (patisiran) or antisense oligonucleotides (inotersen); and 2) prevent dissociation of the TTR tetramer through small molecule TTR stabilizers (tafamidis). Both approaches have yielded positive results in phase 3 studies of hATTR amyloidosis with polyneuropathy 25,26 and tafamidis has also shown benefit in patients with ATTR amyloidosis with cardiomyopathy, 27 with studies underway for patisiran 28 and inotersen. 29 For patients with hATTR amyloidosis to derive maximal benefit from the available therapies, early identification of patients remains essential.
Previous studies of V122I carriers have captured patients late in their disease course and/or looked at a limited set of pre-defined outcomes. 30 The main strength of this study is its size and prospective nature. Moreover, we identify treatable clinical manifestations associated with carrying a variant common in individuals of African ancestry, who are historically understudied in genetic research. 31,32 However, this study must be interpreted in the context of potential limitations. Analyses in the UK Biobank were performed using inpatient hospital ICD10 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.10.20219675 doi: medRxiv preprint diagnosis codes and do not capture outpatient or self-reported diagnoses. Therefore, it is possible our analyses missed additional hATTR amyloidosis manifestations present in participants but not captured in the hospital ICD10 diagnosis codes. Effect estimates of the association between the V122I variant and polyneuropathy were heterogeneous between the 3 biobanks (OR: UK Biobank = 6.4; Penn Medicine Biobank = 1.6; Million Veteran Program = 1.5). This is likely due to differences in the collection methods of ICD diagnosis codes: in the UK Biobank, diagnoses were from inpatient hospital records, whereas in the Penn Medicine Biobank and the Million Veteran Program they include both inpatient and outpatient diagnoses.
Indeed, the rate of polyneuropathy in non-V122I carriers is lower in the UK Biobank, suggesting the higher odds ratio in this population may be due to the method of ICD10 diagnosis collection.
Additional limitations include that UK Biobank participants are healthier on average than the general UK population. 33 Also, despite the large sample size, the number of V122I carriers with common hATTR amyloidosis manifestations in the UK Biobank is small (n = 27), limiting statistical power to detect significant associations. Furthermore, V122I carriers in the UK Biobank were on average 60 years of age at censoring (range: 42-79 years), which is younger than the age range during which V122I hATTR amyloidosis typically presents. 30 We hypothesize that this younger age may be a reason that the well-known cardiac manifestations of hATTR amyloidosis are not as enriched as expected based on previous reports. Lastly, while 4 common hATTR amyloidosis manifestations were found to be enriched in V122I carriers, not all diagnoses of cardiomyopathy, CTS, HF, and polyneuropathy can be attributed to hATTR All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.10.20219675 doi: medRxiv preprint amyloidosis since these are commonly seen with other diseases as well as in an idiopathic manner.
In a large unbiased study of 1 of the most common pathogenic TTR mutations, the V122I All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 13, 2020. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 13, 2020. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.10.20219675 doi: medRxiv preprint 25    All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

Figure 1
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Figure 2
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Figure 3
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The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.10.20219675 doi: medRxiv preprint All UK Biobank study participants underwent a baseline study assessment where covariates including age, sex, body mass index, self-reported diabetes diagnosis, self-reported hypertension diagnosis, and smoking status were collected. Additionally, participants consented to access to their NHS hospital records, which were used to obtain primary and secondary International Statistical Classification of Diseases and Related Health Problems, 10th Revision (ICD10) disease diagnosis codes. Diagnosis codes were collected prospectively from inpatient NHS visits during study follow-up and retrospectively from 1996 to baseline visit.

Penn Medicine Biobank
The Penn Medicine Biobank currently includes over 60 000 participants enrolled through the

Million Veteran Program
The Million Veteran Program is a large multi-ethnic cohort within the United States Department of Veterans Affairs (VA). Since 2011, more than 825 000 veterans over age 18 years have been recruited from 63 participating VA medical center facilities across the United States. 7 The Million Veteran Program biobank incorporates data from biospecimens, baseline and lifestyle surveys, and EHRs, including clinical laboratory measurements, diagnostic imaging reports, diagnosis, and procedure codes and vital status. Million Veteran Program research protoco ls All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. which share >95% common content. Genotypic data were imputed to the UK10K haplotype, 1000 Genomes phase 3, and Haplotype Reference Consortium reference panels using SHAPEIT3 10 for phasing and IMPUTE4 3 for imputation. The V122I variant was directly genotyped on both arrays (info score = 1) and passed all quality control metrics (Hardy-Weinberg equilibrium P = 7.2×10 -5 , genotyping call rate = 100%).

Phenome-Wide Association Analysis and Replication
Phenome-wide association analysis was performed in PLINK (v2.0) 5 using logistic regression with the "firth-fallback" option, which runs Firth regression 11 when logistic regression fails due to a rare outcome. Analyses were performed using the REVEAL/SciDB translational analytics platform from Paradigm4. The significant association between the V122I variant and polyneuropathy diagnosis was replicated in the Penn Medicine Biobank and the Million Veteran Program. In the Penn Medicine Biobank, polyneuropathy was defined as the assignment of the All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.10.20219675 doi: medRxiv preprint ICD9 diagnosis code 357 or ICD10 diagnosis codes of G62 or G63 on two or more separate dates. Association was assessed using logistic regression, controlling for age, sex, and the first 5 principal components. In the Million Veteran Program, polyneuropathy was defined as a diagnosis of "G62," including child codes. Association was assessed using logistic regression, controlling for age, sex, and the first 10 principal components of genetic ancestry in 82 362 unrelated Million Veteran Program participants of African ancestry. and survminer packages (v. 0.4.3). All analyses were controlled for known confounders including age, sex, smoking status, and genetic ancestry via 10 genetic principal components. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

Assessment of Common hATTR Amyloidosis Manifestations in V122I Carriers
The copyright holder for this this version posted November 13, 2020. Abbreviations: BMI indicates body mass index; PC, principal component; SD, standard deviation.
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