
==== Front
Kidney360
Kidney360
KIDNEY
Kidney360
Kidney360
2641-7650
American Society of Nephrology

38689396
K360-2023-000788
10.34067/KID.0000000000000461
00009
3
Clinical Research
Cystic Kidney Disease
Genetic Analysis of Severe Polycystic Liver Disease in Japan
https://orcid.org/0000-0003-4187-1067
Mizuno Hiroki 1 2 3
https://orcid.org/0000-0002-8283-1507
Besse Whitney 4
https://orcid.org/0000-0001-7326-4988
Sekine Akinari 2 3
https://orcid.org/0009-0008-2935-0430
Long Kelly T. 4
Kurihara Shigekazu 1
https://orcid.org/0000-0002-1494-3291
Oba Yuki 1
https://orcid.org/0000-0003-3264-9691
Yamanouchi Masayuki 1
Hasegawa Eiko 2
https://orcid.org/0000-0003-0825-2512
Suwabe Tatsuya 1
https://orcid.org/0000-0003-2092-4094
Sawa Naoki 1
Ubara Yoshifumi 1
https://orcid.org/0000-0001-5062-4629
Somlo Stefan 4 5
https://orcid.org/0000-0002-0444-101X
Hoshino Junichi 2 6
1 Nephrology Center Toranomon Hospital Kajigaya, Kawasaki, Japan
2 Nephrology Center Toranomon Hospital, Tokyo, Japan
3 Okinaka Memorial Institute for Medical Research, Tokyo, Japan
4 Section of Nephrology, Yale University School of Medicine, New Haven, Connecticut
5 Department of Genetics, Yale School of Medicine, New Haven, Connecticut
6 Department of Nephrology, Tokyo Women's Medical University, Tokyo, Japan
Correspondence: Dr. Hiroki Mizuno or Dr. Whitney Besse, email: hilomiz@yahoo.co.jp or whitney.besse@yale.edu
8 2024
01 5 2024
5 8 11061115
25 10 2023
25 4 2024
Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Society of Nephrology
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Visual Abstract

Key Points

Among patients with severe polycystic liver disease (PLD) (height-adjusted total liver volume of <1800 ml/m), PKD2 variants were found in 34%.

Three patients with PKD1 or PKD2 variants are reported with severe PLD but normal-sized kidneys (hTKV of < 250 ml/m).

Background

Polycystic liver disease (PLD) is present in most patients with autosomal dominant polycystic kidney disease (ADPKD). PLD can alternatively be found with few, if any, kidney cysts as a diagnosis of isolated PLD (autosomal dominant PLD [ADPLD]). Several genes are identified as causative for this spectrum of phenotypes; however, the relative incidence of genetic etiologies among patients with severe PLD is unknown.

Methods

Patients with ADPKD or ADPLD having severe PLD defined as height-adjusted total liver volume (hTLV) >1800 ml/m were recruited. Subsequent clinical care was followed. Genetic analysis was performed using whole exome sequencing.

Results

We enrolled and sequenced 49 patients (38 women, 11 men). Pathogenic or suspected pathogenic variants in polycystic disease genes were found in 44 of 49 patients (90%). The disease gene was PKD1 in 20 of 44 patients (45%), PKD2 in 15 of 44 patients (34%), PRKCSH in 5 of 44 patients (11%), GANAB in 2 of 44 patients (5%), SEC63 in 1 of 44 patients (2%), and ALG8 in 1 of 44 patients (2%). The median hTLV was no different between genetically defined ADPKD and ADPLD groups (4431 [range, 1817–9148] versus 3437 [range, 1860–8211]) ml, P = 0.77), whereas height-adjusted kidney volume was larger as expected in ADPKD than in ADPLD (607 [range, 190–2842] versus 179 [range, 138–234] ml/m, P < 0.01). Of the clinically defined ADPKD patients, 20 of 38 patients (53%) were PKD1, 15 of 38 (39%) were PKD2, and 3 (8%) remained genetically unsolved. Among patients with a pathogenic PKD1 or PKD2 variant, we found three patients with a liver-dominant ADPKD (severe PLD with height-adjusted total kidney volume <250 ml/m).

Conclusions

ADPLD-related genes represent 20% of patients with severe PLD in our cohort. Of those enrolled with ADPKD, we observed a higher frequency of PKD2 carriers than in any previously reported ADPKD cohorts. Although there was no significant difference in the hTLV between patients with PKD1 and PKD2 in this cohort, our data suggest that enrollment on the basis of severe PLD may enrich for patients with PKD2.

ADPKD
cystic kidney
gene expression
polycystic kidney disease
Okinaka Memorial Institute for Medical ResearchHiroki MizunoOkinaka Memorial Institute for Medical ResearchHiroki Mizuno OPEN-ACCESSTRUE
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pmcIntroduction

Polycystic liver disease (PLD) is a clinical term used to describe the phenotype of multiple simple fluid-filled cysts in the liver.1 PLD can occur as an extrarenal manifestation of autosomal dominant polycystic kidney disease (ADPKD) or without meaningful kidney cyst burden known as isolated autosomal dominant PLD (ADPLD, also abbreviated PCLD). The prevalence of ADPKD is approximately 1:1000, while 94% of patients with ADPKD over age 35 years have liver cysts, and the severity and clinical relevance of the liver cysts are highly variable.2,3 Clinically identified ADPLD has an estimated prevalence of 1:10,000–158,000, but mild cases may often be clinically undiagnosed.4–6 Patients with symptomatic PLD are often women for reasons attributed to hormonal effects.7

ADPKD and ADPLD are genetic disorders with related mechanisms. ADPKD is caused by heterozygous genetic variants in the PKD1 or PKD2 genes, which encode the polycystin proteins. PKD1 accounts for approximately 77% of genetically defined patients.8,9 ADPLD is caused by heterozygous genetic variants in any of several disease genes: PRKCSH,10 SEC63,11 ALG8,1 SEC61B,12 ALG9,13 or GANAB.12,14 PRKCSH and SEC63 appear to be the most commonly seen disease genes for ADPLD.4 In many cases, GANAB, ALG8, and ALG9 have kidney cysts instead of, or in addition to, liver cysts1 and may be found to explain patients clinically characterized as mild or atypical ADPKD. These ADPLD-related genes encode the proteins in the endoplasmic reticulum, and the loss of function of these genes causes the reduction of the functional polycystin-1 (PC1) in the kidney and liver, which plays a crucial role in cystogenesis.12,15,16

Clinical studies have been conducted to elucidate the clinical course of PLD17–22; however, gene-based comparisons were not performed in these studies to our knowledge. However, given the more recent realization that several genes—particularly GANAB and ALG8—can manifest with kidney cysts in addition to liver cysts, the underlying genetics in these patients may make relevant distinctions. In contrast to ADPKD kidney cyst severity, ADPKD liver cyst severity appears not to be affected by genotype (PKD1 truncating, PKD1 nontruncating, or PKD2).18,23–25 To characterize and learn from the genotypes capable of and likely to produce severe PLD, we set up a cohort study of patients with ADPKD and ADPLD who have severe PLD defined as a height-adjusted total liver volume (hTLV) >1800 ml/m to conduct genetic analysis and genotype–phenotype correlations. We predefined this threshold based on its use in the literature and data suggesting that near or above this liver size clinical symptoms are seen in the majority of patients.25,26

Method

Study Population

Electronic medical records were reviewed for patients with polycystic liver or polycystic kidney who regularly visited from September 2018 to April 2019 in the nephrology department of Toranomon Hospital or Toranomon Hospital Kajigaya. The inclusion criteria of this cohort were patients over 20 years who had computed tomography (CT) or magnetic resonance imaging showing severe polycystic liver (hTLV >1800 ml/m) with or without polycystic kidneys—defined as having >10 cysts in two kidneys—and who were seen in clinic and able to consent. Study participation was offered to all patients meeting these criteria for whom a family member was not already enrolled, and 49 participants chose to enroll and completed sample collection and written informed consent. This study was approved by the institutional review board in Toranomon Hospital and Toranomon Hospital Kajigaya (2018-1), and adhered to the Declaration of Helsinki.

Genetic Evaluation

DNA samples were extracted at Toranomon Hospital. Whole exome sequencing (WES) was performed at the Yale Center for Genome Analysis and analyzed by authors using validated pipelines previously described.12 Only rare variants with a minor allele frequency of <1×10−4 in the gnomAD database were considered.27,28 Nontruncating variants were adjudicated using American College of Medical Genetics criteria.29 Those patients with PKD1 variant of unknown significance (VUS) were characterized as PKD1 patients if the VUS met at least two of the following four criteria—novel in gnomAD,27 CADD score >20,30 deleterious by metaSVM,31 and REVEL >0.7.32 Calling of copy number variation is limited in research-grade WES. Nonetheless, large heterozygous deletions were implicated if two criteria were met: (1) there was a statistically significant reduction (approximately 50%) in read depth throughout one large exon or more than one contiguous exon in comparison with the same exon in samples sequenced in parallel (same flow cell or batch) and (2) the implicated heterozygous deletion region was devoid of heterozygous variants. Implicated heterozygous deletions were confirmed using genomic quantitative PCR. For each region of interest, the relative allele count for the DNA sample from the patient with the suspected large deletion and that of three controls was evaluated in triplicate. Each sample's average cycle threshold for amplification of genomic regions within the implicated deletion was normalized to the same genomic DNA sample's amplification of an intronic region of GAPDH. Case fold difference was compared with the mean fold difference of the three controls to determine the relative allele count. If the mean relative allele count of technical triplicates was ≤50%, the heterozygous deletion was considered confirmed.

Clinical and Radiological Evaluation

All clinical data were retrieved from the electronic medical records. Baseline demographics, laboratory data, and imaging data were collected on the day when the patients wrote informed consent; however, in case they had already experienced surgical interventions, including partial hepatectomy, percutaneous liver cyst aspiration, or transarterial embolization to liver arteries,33 their most recent data before interventions were collected. The eGFR was calculated by the revised equation for Japanese people.34 TLV and total kidney volume were calculated by a semiautomated program (Synapse Vincent, Fujifilm Medical) by CT scan and evaluated for hTLV and height-adjusted total kidney volume (hTKV). The predicted liver growth rate based on baseline imaging findings was calculated using the following equation,18 and the baseline liver volume was postulated using the formula that calculated the standard liver volume for liver transplantation at the age of 18 years35:((hTLV645)1Age−18−1)×100

Moreover, serial hTLVs from multiple imaging studies were collected if available as part of routine clinical care before PLD-related interventions, and patients were classified based on the imaging classification.18 Clinical data were collected blinded to genetic assessment.

Statistical Analysis

Categorical data were described as number and percent and analyzed by a two-tailed Fisher's exact test as appropriate, whereas continuous variables were described as the median and range and were analyzed by the Wilcoxon rank-sum test. Any P values below 0.05 were considered statistically significant. All statistical analyses were conducted by R statistical version 4.2.3 (R Foundation for Statistical Computing, Vienna, Austria, http://R-project.org/).

Results

Baseline Characteristics of PLD Cohort

We identified 701 charts of patients with ADPKD or ADPLD in our center for which the necessary imaging was available. We enrolled 49 patients of 49 families from among the 141 patients with severe PLD (hTLV >1800 ml/m) in our center (Figure 1). Among the 49 patients, 38 had a clinical diagnosis of ADPKD and 11 had a diagnosis of ADPLD (Table 1). The median age was 57 years (range, 36–76), and 38 patients (78%) were female. The median hTLV and hTKV were 4330 (range, 1817–9148) ml/m and 415 (range, 57–2842) ml/m, respectively. Surgical interventions were conducted in 39 patients (89%) (Supplemental Table 1). No patients underwent liver transplantation.

Figure 1 Toranomon hospital polycystic kidney and liver population and patient enrollment flowchart. All patients with severe PLD (hTLV >1800 ml/m) who visited our clinic with the indicated patient phenotype subsets were offered enrollment, and the subset indicated in green text consented and were thus enrolled to participate. Enrollment and clinical indication of ADPKD versus ADPLD were carried out before genetic testing. ADPKD, autosomal dominant polycystic kidney disease; ADPLD, autosomal dominant polycystic liver disease; hTLV, height-adjusted total liver volume; PKD, polycystic kidney disease; PLD, polycystic liver disease.

Table 1 Clinical and genetic data of severe polycystic liver disease cohort

No	Sex	Age at Diagnosis, yr	Family History	Age, yr	hTKV, ml/m	hTLV, ml/m	Clinical Diagnosis	Gene	Variant	
1	F	29	Yes	36	331	6510	ADPKD	PKD1	p.Trp4012X	
2	F	36	Yes	50	360	2974	ADPKD	PKD1	p.Ala1315Pro (VUS)	
3	F	41	Yes	59	572	4825	ADPKD	PKD1	p.Leu727Pro (LP)	
4	M	40	Unk.	76	691	9148	ADPKD	PKD1	p.Arg799Pro (VUS)	
5	M	38	Yes	60	2842	6050	ADPKD	PKD1	p.Thr3135Met (LP)	
6	M	44	Yes	53	2414	6079	ADPKD	PKD1	p.Gln2243X	
7	F	51	Yes	61	1103	2198	ADPKD	PKD1	p.Val878Profs×22	
8	F	30	Yes	51	586	5763	ADPKD	PKD1	c.11017-10C>A	
9	F	41	Yes	56	846	7043	ADPKD	PKD1	p.Gln1068X	
10	F	44	Yes	54	618	5347	ADPKD	PKD1	p.Arg4150Alafs×48	
11	M	46	Unk.	63	2255	8518	ADPKD	PKD1	p.Gln1116X	
12	F	40	Yes	68	1098	6166	ADPKD	PKD1	p.Gln227X	
13	F	29	Yes	46	415	3389	ADPKD	PKD1	p.Ile985_Val991del	
14	F	55	Yes	76	1065	3112	ADPKD	PKD1	p.Asn1269Leufs×31	
15	F	36	Yes	43	816	4759	ADPKD	PKD1	p.Arg2163X	
16	F	40	Yes	62	607	5996	ADPKD	PKD1	p.Phe2133del (LP)	
17	F	41	Yes	46	1102	3910	ADPKD	PKD1	p.Ser1915X	
18	F	46	Yes	59	190	2209	ADPKD	PKD1	p.Tyr1441Cys (VUS)	
19	F	44	Yes	50	435	3055	ADPKD	PKD1	p.Thr3049Ile (VUS)	
20	F	47	Yes	68	690	1912	ADPKD	PKD1	p.Trp4012X	
21	F	42	Yes	57	930	3714	ADPKD	PKD2	p.Arg872X	
22	F	34	Yes	43	386	4162	ADPKD	PKD2	p.Gly143Alafs×90	
23	M	52	Yes	66	1194	4553	ADPKD	PKD2	p.Arg322Gln (P)	
24	F	49	Yes	49	202	1817	ADPKD	PKD2	p.Arg803X	
25	F	29	No	42	913	4431	ADPKD	PKD2	c.1549-1 G>GCTGT	
26	F	35	Yes	45	294	4700	ADPKD	PKD2	p.Asp339Valfs×36	
27	F	45	Yes	51	315	6051	ADPKD	PKD2	p.Arg654X	
28	M	40	Yes	53	257	3185	ADPKD	PKD2	p.Arg306X	
29	F	30	Yes	50	678	3702	ADPKD	PKD2	p.Cys509X	
30	M	61	Yes	62	657	5360	ADPKD	PKD2	p.Trp293X	
31	F	58	Yes	60	595	4330	ADPKD	PKD2	p.Arg322Gln (P)	
32	F	41	Yes	61	332	2611	ADPKD	PKD2	p.Tyr247X	
33	F	34	No	38	284	5169	ADPKD	PKD2	p.Arg872X	
34	F	41	Yes	57	282	3333	ADPKD	PKD2	p.Gly143Alafs×90	
35	F	37	Yes	50	210	2936	ADPKD	PKD2	p.Arg592X	
36	F	51	Yes	57	234	3437	ADPLD	GANAB	p.Thr851Pro (NM_198334) (VUS)	
37	F	50	Yes	61	162	6318	ADPLD	GANAB	p.Thr851Pro (NM_198334) (VUS)	
38	M	45	Yes	62	170	7454	ADPLD	PRKCSH	Exon 1 and 2 del.	
39	F	51	No	55	198	3162	ADPLD	PRKCSH	p.Glu125Valfs×21	
40	F	44	Unk.	51	179	1960	ADPLD	PRKCSH	c.1431+1G>A	
41	M	60	No	71	208	8211	ADPLD	PRKCSH	p.Glu125Valfs×21	
42	F	Young	Yes	72	138	2394	ADPLD	PRKCSH	p.Tyr423X	
43	F	50	Yes	57	166	3387	ADPLD	SEC63	p.Thr469Leufs×7	
44	F	45	Unk.	48	225	3527	ADPLD	ALG8	Exon 1 deletion	
45	F	37	Yes	38	280	6377	ADPKD	Unsolved	n/a	
46	F	40s	Yes	71	995	6020	ADPKD	Unsolved	n/a	
47	F	66	Unk.	67	162	2321	ADPLD	Unsolved	n/a	
48	M	38	Unk.	49	967	6544	ADPKD	Unsolved	n/a	
49	M	Unknown	No	75	57	3251	ADPLD	Unsolved	n/a	
ADPKD, autosomal dominant polycystic kidney disease; ADPLD, autosomal dominant polycystic liver disease; hTKV, height-adjusted total kidney volume; hTLV, height-adjusted total liver volume; LP, likely pathogenic; n/a, not available; P, pathogenic; PKD, polycystic kidney disease; PLD, polycystic liver disease; VUS, variant of unknown significance.

Genetic Analysis

We carried out WES on a research basis for all members in the enrolled cohort. Genetic findings are shown in Figure 2 and Table 1. We found pathogenic or suspected pathogenic variants in 44 of 49 patients (90%). The disease gene was PKD1 or PKD2 in 35 of 44 (80%) patients, whereas other ADPLD-related genes were found in 9 of 44 (20%) patients. Of those with PKD1 or PKD2 variants, an unusually high percentage of 15 of 35 (43%) patients contained their pathogenic variant in PKD2 (Tables 1 and 2). Of the PKD1 variants, 11 of 20 were truncating, and all 20 were unique from each other (Table 1). Of the PKD2 variants, 13 of 15 were truncating. There were two patients with the PKD2 p.Arg872X, two with PKD2 p.Arg322Gln, and two with PKD2 p.Gly143Alafs. We used the relatedness2 tool from vcftools36 to ensure that no close familiar relationships were present in the cohort. No close (less than or equal to third degree) familial relationship was noted between these PKD2 patients, but one relationship was found between the two GANAB patients. Only one patient had the PKD2 p.Arg803X variant reported to be a founder mutation in a Taiwanese population.37 One of the PKD2 patients (patient 23) had both the PKD2 p.Arg322Gln variant (“highly likely pathogenic” with multiple references in https://pkdb.mayo.edu/variants) and a rare VUS in PKD1, p.Gln2376Arg.

Figure 2 Disease gene for patients with severe PLD (height-adjusted total liver >1800 ml/m) in this study.

Table 2 The relative incidence of PKD1/PKD2 variant carriers in published large cohorts of autosomal dominant polycystic kidney disease

Reference	45	46	47	48	25	49	50	51	52	53	This Study	This Study ADPKD Subset	
Year	2006	2009	2014	2016	2016	2018	2022	2022	2022	2023	2023	2023	
Country	United States	Canada	United States	France	United States	United States	Korea	Japan	Japan	Japan	Japan	Japan	
Number of samples, n	241	484	447	913	434	770	162	118	129	436	49	38	
PKD1, n (%)	153 (64)	367 (76)	362 (81)	678 (74)	362 (83)	656 (85)	116 (72)	90 (76)	99 (77)	273 (63)	20 (41)	20 (53)	
PKD2, n (%)	31 (13)	117 (24)	60 (13)	172 (19)	72 (17)	93 (12)	25 (15)	14 (12)	21 (16)	90 (21)	15 (31)	15 (39)	
Non-PKD1, PKD2, n (%)	0	0	0	0	0	0	0	0	0	3 (1)	9 (18)	0	
Unsolved, n (%)	56 (23)	0 (0.0)	25 (6)	63 (7)	0 (0.0)	21 (3)	21 (13)	14 (12)	9 (7.0)	70 (16)	5 (10)	3 (8)	
ADPKD, autosomal dominant polycystic kidney disease; PKD, polycystic kidney disease.

Twenty percent of this cohort—ascertained based on severe PLD—had pathogenic variants in non-PKD1/PKD2 genes (Figure 2 and Table 1). PRKCSH contained the pathogenic variant in 5 of 44 (11.4%) patients, GANAB in 2 of 44 (5%) patients, SEC63 in 1 of 44 (2%) patients, and ALG8 in 1 of 44 (2%) patients. Relatedness analysis found an unexpected relationship between patient 36 and patient 37, where the patients appear to be either third-degree or fourth-degree relatives. This was the only close familial relationship detected in the cohort. Two of the called variants (one in PRKCSH, one in ALG8) were large heterozygous deletions found from whole exome data and validated with genomic quantitative PCR for this study.

Clinical Characteristics between Genotypes

We next compared clinical characteristics between those non-PKD1/PKD2 patients (n=9) and the PKD1/PKD2 patients (n=35). We found no significant difference in hTLVs, which were 3437 (range, 1960–8211) ml/m and 4431 (range, 1817–9148) ml/m, respectively, P value = 0.77. The number and types of interventions pursued for symptomatic management of PLD management were similar (Supplemental Tables 1 and 2). All of the patients with GANAB, PRKCSH, SEC63, or ALG8 variants were indicated by their clinician as having PLD without polycystic kidney disease (PKD) (hTKV <250 ml/m), although several have a small number of kidney cysts (Table 1). The non-PKD1/PKD2 group's mean hTKV was 179 (range, 138–234) ml/m compared with 607 (range, 190–2842) ml/m in the PKD1 or PKD2 group (P value < 0.01), and only one PKD1 and two PKD2 patients had hTKV <250 ml/m (Figure 3 and Supplemental Table 2). There was no significant correlation between hTLV and hTKV in each group (Figure 3).

Figure 3 hTLV and hTKV by genotype. hTKV and hTLV measurements were calculated from the initial MRI or CT images available at enrollment. Each patient provides one datapoint. Datapoints are on separate plots based on disease genes determined after WES analysis. There was no significant correlation between hTLV and hTKV in each group; 0.37 (95% CI, −0.08 to 0.69) in PKD1 patients, 0.28 (95% CI, −0.27 to 0.70) in PKD2 patients, and 0.05 (95% CI, −0.64 to 0.69) in non-PKD1/PKD2 patients. A threshold of hTKV <250 ml/m (vertical line) would include all GANAB, PRKCSH, SEC63, or ALG8 patients in this cohort and one PKD1 and two PKD2 patients and may be used to predict whether unsolved patients might be expected to be missed PKD1 or PKD2 variants versus another gene. The horizontal line represents hTLV 1800 ml/m. CI, confidence interval; CT, computed tomography; hTKV, height-adjusted total kidney volume; WES, whole exome sequencing.

Clinical Comparison between PKD1 and PKD2 Patients

Compared with those with the PKD1 variant, patients with PKD2 had more mild kidney parameters including smaller hTKV (332 [range, 202–1194] ml/m, 691 [range, 190–2842] ml/m, P value = 0.02) and lower serum creatinine level (0.70 [range, 0.50–1.80] mg/dl, 1.08 [range, 0.55–13.80] mg/dl, P value = 0.01). Liver parameters were similar between these two groups. hTLV was slightly higher in PKD1 patients than in PKD2 patients, but this difference was not significant (NS) (5086 [1912–9148] ml/m, 4162 [1817–6051] ml/m, P = 0.16) and may be influenced by the slightly later age at enrollment of PKD1 patients. The one exception is that liver function test alanine aminotransferase was higher in patients with PKD2 (18 [10–75] versus 13 [6–68], P=0.01) (Table 1 and Supplemental Table 3).

Assessment of Liver Size and Growth Rate with Age

There was no significant difference between the predicted growth rate in patients with GANAB, PRKCSH, SEC63, or ALG8 pathogenic variants compared with those with PKD1 or PKD2 variants (4.6 [range, 2.5–5.8]%/yr versus 5.2 [range 2.3–14.0]%/yr) (P=0.17) (Figure 4 and Supplemental Table 2). As it has been reported that the trajectory of hTLV does not always progress linearly,25 we analyzed the series of CT images of patients where imaging was available at multiple time points without procedural interventions for assessment of the progression of hTLV (Figure 5). The serial volumetry data were obtained from 21 patients, representing PKD1, PKD2, and PRKCSH patients. The median age available for serial data was 59 years (range, 49–76), and 14 patients (67%) were female (Supplemental Table 4).

Figure 4 Relationship between hTLV and age at imaging. hTLV versus age plotted separately depending on disease gene. The oblique line represents 5% per year of hTLV growth.

Figure 5 Serial measurements of hTLV. hTLV values for patients with at least two imaging studies suitable for hTLV calculation without intervening interventions. Red indicates female, and blue indicates male.

PLD-Dominant ADPKD Cases

Among the PKD1 or PKD2 patients, we noted one PKD1 patient and two PKD2 patients with hTKV <250 ml/m at the age of 49–59 years despite severe PLD—in other words, they had a PLD-dominant phenotype with unusually mild PKD albeit still meeting ADPKD clinical diagnosis. Each of these patients were female. The PKD1 patient (patient 18), attributed to a PKD1 p.Tyr1441Cys variant, was diagnosed with PLD when she was 46 years and had numerous small cysts in the kidney, and at age 59 years, hTKV was 190 ml/m whereas hTLV was 2209 ml/m (Figure 6A). Her kidney function was within normal range, but she had hypertension. Patient 35, carrying the pathogenic PKD2 p.Arg592X variant, was diagnosed with PLD when she was 37 years, and imaging at age 50 years showed hKTV was 210 ml/m while her hTLV was 2936 ml/m. She had two childbirths with two gravidae and never used oral contraceptives. Her hTLV was symptomatic; therefore, a transarterial embolization of liver arteries was conducted at age 50 years (Figure 6B). Patient 24, carrying the pathogenic PKD2 p.Arg803X variant, was diagnosed with PLD when she was 49 years old, and imaging at that time showed hTKV of 202 ml/m and hTLV of 1817 ml/m. She had experienced three child births with three gravidae and never used oral contraceptives.

Figure 6 Patients of interest attributable to PKD1 or PKD2 variants. (A) Patient 18, PKD1 variant (p.Tyr1441Cys). A 46-year-old woman with hTKV of 190 ml/m and hTLV of 2209 ml/m. Her kidney function is within normal range, although she has hypertension. (B) Patient 35, PKD2 variant (p.Arg592X). A 50-year-old woman with hTKV of 210 ml/m and hTLV of 2936 ml/m. (C) Patient 19, PKD1 variant (p.Thr3049Ile)—GPS-cleavage site. A 50-year-old woman with hKTV of 435 ml/m and hTLV of 3055 ml/m.

First Report of Patient with a Variant in G-Protein–Coupled Receptor Proteolytic Site Cleavage Site

The mature functional PC1 is known to exist as a cleaved protein. In vitro studies and mouse models have been made to study the consequence of missense variant at the G-protein–coupled receptor proteolytic site cleavage site (orthologs of human p.Leu3048His and p.Thr3049Val).38 The mouse model of p.Leu3048His was embryonically lethal, and despite sufficient expression of uncleaved PC1 in the kidney, the phenotype upon conditional inactivation was no different than truncating models with comparable induction.39 In contrast, mice with the ortholog of p.Thr3049Val studied by a different group survived embryogenesis, suggesting a hypomorphic consequence.39,40 One of our patients carries a PKD1 p.Thr3049Ile variant (Table 1). This is the first human case reported with a GPS cleavage site variant to our knowledge. There are no patients with variants affecting p.Leu3048 or p.Thr3049 in the Mayo database,41 and incomplete information is available for reports of p.Leu3048Phe, p.Leu3048Arg, and p.Thr3049Ile in ClinVar42 and Genome Browser.43 This female patient was diagnosed at age 44 years and had hTKV of 435 ml/m and hTLV of 3055 ml/m at age 50 years (patient 19) (Table 1). Her increased hTKV appeared mainly attributable to a smaller number of large cysts, and renal calculi were notable (Figure 6C). Based on what is known about this amino acid position and the lack of alternative pathogenic variants in this patient with a definitive clinical diagnosis of ADPKD, we are confident that this variant is pathogenic to her disease. Her kidney disease could be classified as mild (Mayo Classification class 1B44) as her serum creatinine was only 0.55 mg/dl at age 50 years. Yet, the phenotype appears consistent with that expected from a fully penetrant pathogenic allele. Her PLD was symptomatic, and she underwent transarterial embolization of a liver artery at age 50 years.

Discussion

In this genetic analysis for severe PLD, we found that most cases occurred in the context of ADPKD and that the clinical diagnosis of ADPKD was specific to PKD1 or PKD2 genotype. Nonetheless, genetically confirmed ADPLD—a disease thought to be much rarer than ADPKD—was the cause of 20% of genetically explained severe PLD in our nephrology center. This demonstrates that ADPLD is an important contributor to the population burden of severe PLD. Factors to consider which may affect the composition of our cohort are anything affecting patient motivation to participate—although the participation rate was the same approximately 34% from both the ADPKD and ADPLD phenotype groups, indication for abdominal imaging suitable for hTLV calculation, and indication for referral to our center that provides consideration of interventional therapies. Nonetheless, this cohort is unique in its inclusion of ADPLD patients, genetic information, and many interventions.

A striking finding from our genetic analysis is the apparent enrichment of PKD2 patients in this uniquely selected cohort compared with previously published ADPKD cohorts from Japan or other countries. In this study, PKD2 variants were found in 39% of the clinically defined ADPKD patients. PKD2 typically accounts for 12%–24%, whereas those with PKD1 account for 63%–85% 25,45–49 (Table 2). In Asian cohorts, the rates of the PKD1 and PKD2 variants are similar to those of previous studies.50–53 This cohort was selected before any genetic assessment and without considering kidney status. Although PLD in PKD2 patients was no more severe than in PKD1 patients—consistent with published findings in other cohorts—one explanation of our prevalent PKD2 patients could be that severe PLD is more prevalent among PKD2 patients, thus enriching this genotype in ADPKD cohorts defined based on severe PLD.25

Although not all kidneys with cysts in this cohort were enlarged, variants in GANAB, ALG8, ALG9, and DNAJB11 may cause phenotypes ranging from clinically suspected ADPKD to ADPLD, although they did not meet accepted clinical diagnostic criteria for ADPKD. In these patients, hTKV was consistently <250 ml/m, whereas this was true for only 3 of 35 patients with a PKD1 or PKD2 variant (PLD-dominant ADPKD patients). This suggests that hTKV >250 ml/m could be a logical threshold above which suspicion should be high for PKD1 or PKD2 variants.

In this cohort, we found a novel PKD1 p.Tyr1441Cys missense variant from a patient with severe PLD but relatively mild PKD. This variant is predicted to affect a single amino acid in one of the PKD domains of the extracellular N terminus of PC1. It will be interesting whether future cases with this variant identified in the future or the patient's family also have mild kidney disease. Our patient with the novel GPS cleavage site variant PKD1 p.Tyr3049Ile has a less mild kidney disease, with hTKV of >400 ml/m in addition to severe PLD, suggesting that this novel GPS cleavage site variant has a fully penetrant pathogenic consequence in humans, a finding that provides clarity to the mouse model investigations that posed this question.38,39

The female predominance described in clinically apparent PLD is indeed confirmed in our cohort similar with percentages similar to other cohorts. In our cohort of severe PLD, 80% of the 38 patients with ADPKD, and 73% of the 11 patients with ADPLD, are female. This is similar to 83% of the 101 ADPKD patients with severe PLD described by Chebib et al.25 and the 76% of 201 ADPLD patients described by Schönauer et al.54 Compared with the Schonauer cohort, our cohort has higher mean hTLV despite slightly lower mean age at assessment.

The strength of this study is that we recruited based on severe PLD without regard to ADPKD status and evaluated both ADPKD- and ADPLD-related genes without prior clinical information. In being the first study to our knowledge to take this approach, we define the genetic basis of severe PLD, although the number of patients is small and from only a subset of the eligible population in two referral centers in Japan. Furthermore, although we propose an intriguing enrichment of PKD2 patients in this cohort, we interpret this enrichment compared with ratios (PKD1:PKD2:Unsolved) in historical cohorts and not our study population because genetic data for this “true reference” ADPKD population are not available.

In conclusion, we describe PKD1, PKD2, PRKCSH, GANAB, SEC63, and ALG8 as disease genes contributing to the burden of severe PLD in a Japanese population. We confirm prior findings that liver cyst severity may be independent of kidney cyst severity and that genotype does not predict severity. We find an apparent enrichment in PKD2 patients and propose that severe PLD may be more prevalent in PKD2-related ADPKD.

Supplementary Material

Disclosures

Disclosure forms, as provided by each author, are available with the online version of the article at http://links.lww.com/KN9/A513.

Funding

H. Mizuno: Okinaka Memorial Institute for Medical Research. W. Besse: NIH/NIDDK K08DK119642. Yale Center for Mendelian Genomics (CMG): NIH M#UM1HG006504-05. J Hoshino:MHLW Research Program on Intractable Kidney Disease (JPMH23FC1048) Intractable Hepatobiliary Disease (JPMH20FC1023), and JSPS KAKENHI 18K08227 and 24K11440.

Author Contributions

Conceptualization: Whitney Besse, Junichi Hoshino, Hiroki Mizuno, Akinari Sekine, Stefan Somlo, Yoshifumi Ubara.

Data curation: Whitney Besse, Eiko Hasegawa, Junichi Hoshino, Shigekazu Kurihara, Hiroki Mizuno, Yuki Oba, Naoki Sawa, Akinari Sekine, Tatsuya Suwabe, Yoshifumi Ubara, Masayuki Yamanouchi.

Formal analysis: Whitney Besse, Hiroki Mizuno, Akinari Sekine.

Funding acquisition: Whitney Besse.

Investigation: Whitney Besse, Junichi Hoshino, Kelly T. Long, Hiroki Mizuno.

Methodology: Whitney Besse, Junichi Hoshino, Hiroki Mizuno.

Project administration: Whitney Besse, Hiroki Mizuno.

Resources: Whitney Besse, Hiroki Mizuno.

Software: Whitney Besse, Hiroki Mizuno.

Supervision: Junichi Hoshino, Stefan Somlo.

Validation: Whitney Besse, Junichi Hoshino, Hiroki Mizuno.

Visualization: Whitney Besse, Junichi Hoshino, Hiroki Mizuno, Akinari Sekine.

Writing original draft: Whitney Besse, Junichi Hoshino, Hiroki Mizuno.

Writing review & editing: Whitney Besse, Eiko Hasegawa, Junichi Hoshino, Shigekazu Kurihara, Hiroki Mizuno, Yuki Oba, Naoki Sawa, Akinari Sekine, Stefan Somlo, Tatsuya Suwabe, Yoshifumi Ubara, Masayuki Yamanouchi.

Data Sharing Statement

All data are included in the manuscript and/or supporting information.

Supplemental Material

This article contains the following supplemental material online at http://links.lww.com/KN9/A512.

Supplemental Table 1. History of interventions to polycystic liver cyst.

Supplemental Table 2. Clinical characteristics of PKD1/PKD2 variants group and ADPLD-related variants group. ADPLD-related variants group included patients with PRKCSH, SEC63, GANAB, and ALG8. Continuous variables are described by median and range.

Supplemental Table 3. Clinical characteristics of each variant group.

Supplemental Table 4. Longitudinal volumetrics of hTLV before intervention.

H.M. and W.B. contributed equally to this work.
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