van de Laarschot et al. Orphanet J Rare Dis (2020) 15:302 https://doi.org/10.1186/s13023-020-01585-4 RESEARCH Open Access Novel GANAB variants associated with polycystic liver disease Liyanne F. M. van de Laarschot1, René H. M. te Morsche1, Alexander Hoischen2, Hanka Venselaar3, Hennie M. Roelofs1, Wybrich R. Cnossen1, Jesus M. Banales4,5,6, Ronald Roepman2† and Joost P. H. Drenth1*† Abstract Background: Polycystic liver disease (PLD) is an inherited disorder characterized by numerous cysts in the liver. Auto- somal dominant polycystic kidney and liver disease (ADPKD and ADPLD, respectively) have been linked to pathogenic GANAB variants. GANAB encodes the α-subunit of glucosidase II (GIIα). Here, we report the identifcation of novel GANAB variants in an international cohort of patients with the primary phenotype of PLD using molecular inversion probe analysis. Results: Five novel GANAB variants were identifed in a cohort of 625 patients with ADPKD or ADPLD. In silico analysis revealed that these variants are likely to afect functionally important domains of glucosidase II α-subunit. Missense variant c.1835G>C p.(Arg612Pro) was predicted to disrupt the structure of the active site of the protein, likely reduc- ing its activity. Frameshift variant c.687delT p.(Asp229Glufs*60) introduces a premature termination codon predicted to have no activity. Two nonsense variants (c.2509C>T; p.(Arg837*), and c.2656C>T; p.(Arg886*)) and splice variant c.2002 1G>C, which causes aberrant pre-mRNA splicing and afecting RNA processing, result in truncated proteins and are+ predicted to cause abnormal binding of α- and β-subunits of glucosidase II, thus afecting its enzymatic activ- ity. Analysis of glucosidase II subunits in cell lines shows expression of a truncated GIIα protein in cells with c.687delT, c.2509C>T, c.2656C>T, and c.2002 1G>C variants. Incomplete colocalization of the subunits was present in cells with c.687delT or c.2002 1G>C variants.+ Other variants showed normal distribution of GIIα protein. + Conclusions: We identifed fve novel GANAB variants associated with PLD in both ADPKD and ADPLD patients sup- porting a common pathway in cystogenesis. These variants may lead to decreased or complete loss of enzymatic activity of glucosidase II which makes GANAB a candidate gene to be screened in patients with an unknown genetic background. Keywords: Polycystic liver disease, Glucosidase II, Molecular inversion probe analysis, Liver cysts, Cholangiocytes Background kidney cysts, but renal function remains preserved [1, Development of numerous liver cysts is the primary 2]. On the contrary, kidney cysts are the dominant fea- phenotype of autosomal dominant polycystic liver dis- ture of patients with autosomal dominant polycystic kid- ease (ADPLD). ADPLD patients may present with few ney disease (ADPKD), and the majority of these patients develop concurrent liver cysts [3]. Almost all ADPKD patients harbour gene mutations in polycystic kidney dis- *Correspondence: [email protected] ease 1 (PKD1) or polycystic kidney disease 2 (PKD2) [4]. †Ronald Roepman and Joost P. H. Drenth should be considered joint senior author Te genetic landscape in ADPLD is more diverse as, to 1 Department of Gastroenterology and Hepatology, Institute date, mutations in at least seven genes explain collectively for Molecular Life Sciences, Radboud University Medical Center, P.O. only 25–30% of the genetic spectrum of ADPLD [2, 5]. Box 9101, 6500 HB Nijmegen, The Netherlands Full list of author information is available at the end of the article Although ADPLD and ADPKD are two distinct genetic © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. van de Laarschot et al. Orphanet J Rare Dis (2020) 15:302 Page 2 of 11 disorders, they share PLD as a major phenotypic fea- explore the incidence of GANAB mutations in a large, ture. Recent evidence suggests that the involved proteins independent cohort of patients with PLD as dominant interact, with decreased levels of functional polycystin-1 phenotype, and to evaluate the efect of GANAB muta- (PC1), encoded by PKD1, as the central element for cyst tions on protein structure and glucosidase II subunit development [6, 7]. binding. With the exception of LRP5 (a transmembrane protein part of the LRP5/LRP6/Frizzled co-receptor complex [8, 9]) all genes involved in ADPLD encode proteins involved Results in endoplasmic reticulum trafcking and quality control Patient characteristics of glycoproteins. Tese genes include PRKCSH, SEC63, For our molecular inversion probe (MIP) analysis we GANAB, ALG8, SEC61B, DNAJB11, and ALG9 [10]. Cen- included a total of 625 patients with ADPKD or ADPLD. tral to the pathomechanism of ADPLD is glucosidase II, Patients were referred to our center for genetic analy- an endoplasmic reticulum (ER) resident N-linked glycan- sis or treatment of PLD by hospitals from the Nether- processing enzyme. Glucosidase II (GII) is a complex of lands, Belgium, Spain, and Denmark. All patients had catalytic α-subunit (GIIα), encoded by GANAB, and a the primary phenotype of polycystic liver disease (PLD). regulatory β-subunit (GIIβ), encoded by PRKCSH. Col- Patients were diagnosed with ADPKD or ADPLD based lectively this contributes to the localization and enzy- on clinical presentation. 90 patients were diagnosed with matic activity of GIIα [11]. Glucosidase II catalyzes the ADPKD, and 535 patients with ADPLD. Of the 625 ana- two-step hydrolysis at α1,3-linked glucose–glucose and lysed patients, 17 families were included that consisted glucose–mannose residues of high-mannose-type gly- of 62 family members. Patients had not been genetically cans to generate a quality control protein tag on glyco- screened previously. proteins that is recognized by ER chaperones [12]. As Of the eight patients with likely pathogenic heterozy- such, it acts as a major partner in glycoprotein processing gous GANAB variants, seven patients presented with and quality control in the ER [13]. ADPLD and three patients had 1–3 kidney cysts, while in Te concept that genetic defects disrupting difer- the remaining patients no kidney cysts could be detected. ent subunits of a protein complex result in a very simi- One patient was diagnosed with ADPKD. Te hepatic lar phenotype (“guilt by association”) is well-accepted phenotype did not difer between patients afected by [14]. While mutations in GIIβ have been associated with ADPLD or ADPKD. Six out of eight patients were female ADPLD, the phenotypic picture for GIIα is less clear. and the mean age was 56 (range 31–79) years. Clinical Most studies have linked GANAB to ADPKD and renal presentation of patients with GANAB variants can be cysts were present in most patients [15]. We set out to found in Table 1. Table 1 Clinical presentation of kidney and liver disease in 8 afected individuals with GANAB variants Family 11-0741 Unrelated individuals 8951 9087 8380 11726 11475 11716 8700 12241 Gender Female Female Male Female Male Female Female Female Diagnosis ADPLD ADPLD ADPLD ADPLD ADPLD ADPKD ADPLD ADPLD Mutation (RefSeq NM_198335) Chromo- g.62400926TG>G g.62400926TG>G g.62393841C>T g.62394111C>T g.62396665G>C g.62414056delTAG g.62397125G>C g.62397125G>C some CGG position (Hg19) cDNA c.687delT c.687delT c.2656C>T c.2509C>T c.2002 1G>C c.11_16delTAG CGG c.1835G>C c.1835G>C + change Amino acid p.Asp- p.Asp- p.Arg886* p.Arg837* Splicing variant p.Val4_Ala5del p.Arg612Pro p.Arg612Pro change 229Glufs*60 229Glufs*60 Radiological presentation Type CT Ultrasound CT CT n/a MRI CT CT Liver cysts > 20 cysts multiple cysts 15 cysts > 20 cysts n/a > 20 cysts > 10 cysts > 30 cysts kidney No No 2 bilateral cysts No n/a > 20 bilateral cysts 3 cysts in left 1 cyst in left kidney cysts kidney ADPKD autosomal dominant polycystic kidney disease, ADPLD autosomal dominant polycystic liver disease, CT computed tomography, MRI magnetic resonance imaging, n/a not available for review van de Laarschot et al. Orphanet J Rare Dis (2020) 15:302 Page 3 of 11 Identifcation of GANAB variants in PLD-afected Segregation analysis of GANAB variants individuals and families To bolster the causal relationship between GANAB vari- MIP analysis identifed 38 variants in 32 patients, ants identifed by MIP analysis and clinical presentation of which 13 heterozygous GANAB variants are pos- of PLD, we tested DNA of family members for GANAB sibly pathogenic. We identifed one frameshift, one variants. We confrmed that frameshift variant c.687delT in-frame deletion, one splice site, two nonsense, and co-segregated with the disease in family 11-0741. Tis eight missense variants that were all validated by ADPLD family presented with multiple liver cysts, with- Sanger sequencing (Fig. 1, Additional File 1). Nota- out renal cysts which suggests that the variant is likely bly, none of these variants have been reported before pathogenic.