Exome sequencing reveals IFT172 variants in patients with non-syndromic cholestatic liver disease

Abstract

Background and aim

Gene defects contribute to the aetiology of intrahepatic cholestasis. We aimed to explore the outcome of whole-exome sequencing (WES) in a cohort of 51 patients with this diagnosis.

Patients and methods

Both paediatric (n = 33) and adult (n = 18) patients with cholestatic liver disease of unknown aetiology were eligible. WES was used for reassessment of 34 patients (23 children) without diagnostic genotypes in ABCB11, ATP8B1, ABCB4 or JAG1 demonstrable by previous Sanger sequencing, and for primary assessment of additional 17 patients (10 children). Nasopharyngeal swab mRNA was analysed to address variant pathogenicity in two families.

Results

WES revealed biallelic variation in 3 ciliopathy genes (PKHD1, TMEM67 and IFT172) in 4 clinically unrelated index subjects (3 children and 1 adult), heterozygosity for a known variant in PPOX in one adult index subject, and homozygosity for an unreported splice-site variation in F11R in one child. Whereas phenotypes of the index patients with mutated PKHD1, TMEM67, and PPOX corresponded with those elsewhere reported, how F11R variation underlies liver disease remains unclear. Two unrelated patients harboured different novel biallelic variants in IFT172, a gene implicated in short-rib thoracic dysplasia 10 and Bardet-Biedl syndrome 20. One patient, a homozygote for IFT172 rs780205001 c.167A>C p.(Lys56Thr) born to first cousins, had liver disease, interpreted on biopsy aged 4y as glycogen storage disease, followed by adult-onset nephronophthisis at 25y. The other, a compound heterozygote for novel frameshift variant IFT172 NM_015662.3 c.2070del p.(Met690Ilefs*11) and 2 syntenic missense variants IFT172 rs776310391 c.157T>A p.(Phe53Ile) and rs746462745 c.164C>G p.(Thr55Ser), had a severe 8mo cholestatic episode in early infancy, with persisting hyperbilirubinemia and fibrosis on imaging studies at 17y. No patient had skeletal malformations.

Conclusion

Our findings suggest association of IFT172 variants with non-syndromic cholestatic liver disease.

Introduction

Genetic defects contribute to intrahepatic cholestasis in both children and adults [1]. Using a phenotype-based candidate gene approach, patients can be successfully diagnosed by Sanger sequencing; nonetheless, sequencing of a 66-gene cholestasis panel designed by EGL Genetics in 2017 increased the chance of diagnostic success in infants, children, and young adults, replacing single-gene analysis as a primary diagnostic tool [2]. The Mayo Clinic Laboratories currently offer testing for 112 “cholestasis genes” (test ID: CHLGP); even this is likely not exhaustive.

Limitations imposed by panel size can be overcome by focused-exome sequencing (FES) using capture platforms that target ~5000 genes mutated in Mendelian genetic diseases (Mendeliome) [3] or by whole-exome sequencing (WES). WES is still considered a discovery tool rather than a standard diagnostic procedure although its usefulness has been repeatedly proven in paediatric patients and recently also in adults [4]. The main advantage of WES, coverage of most expressed transcripts, opens the way to unexpected diagnoses, with identification of novel genetic disorders [5–10] and gene-phenotype relationships such as liver manifestations of known extrahepatic diseases without or with secondary liver injury. Whereas the main disadvantageous features of WES–need for excessive data processing and higher cost–have already been overcome, one should keep in mind that, as with FES or panel sequencing, even in WES methodology limitations inhere: Not all exons may be captured, sensitivity for structural variations is low, and WES does not include most non-coding regions [11].

In this study we explored the contribution of WES to molecular diagnosis in 51 patients with intrahepatic cholestasis.

Patients and methods

Patients

Patients with cholestatic liver disease or a history of cholestasis of unknown aetiology documented by detailed medical reports from the referring centre were eligible. Cholestasis was defined as conjugated hyperbilirubinemia accompanied by bilirubinostasis in hepatocytes, canaliculi and Kupffer cells in acinar zone 3, and / or by ductular reaction in acinar zone 1 on histopathologic study, or by elevated serum bile salt level exceeding 10 μmol/l. Patients with extrahepatic bile duct obstruction or autoimmune hepatobiliary disease and those fed only parenterally were excluded. Phenotyping was based on clinical reports and accessible histopathology reports provided by the referring centres (see S1 File). The authors had access to information that could identify individual participants during or after data collection. Where possible, samples from first-degree relatives were obtained for segregation studies. Composed identifiers (IDs) used for index subjects in the results section and tables indicate gender (M—male, F–female), patient age in years at specimen receipt, origin (CE—Central Europe, RO–Roma, PA–Australian Pacific, EA–East Asia, ME–Middle East), and unique number indicating order of specimen receipt. All 18 enrolled adult patients, legal guardians of all 33 enrolled children and all 58 available first-degree relatives who provided DNA samples signed written informed consent for genetic testing approved by the relevant institutional human research committees. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institution’s human research committee of IKEM and Thomayer Hospital in Prague, Czech Republic (Docket No. 17-06-18, date June 28, 2017, study period from July 2017 till December 2022).

Thirty-four enrolled index patients (samples collected between December 2006 and February 2021) represented a subset of 109 patients negatively screened by Sanger sequencing since June 2002 (Fig 1). Most of the remaining 75 patients were lost to clinical follow-up; in a few the diagnosis was re-classified as non-genetic disease. Seventeen index patients with clinically uncertain diagnosis due to various extrahepatic comorbidities such as kidney disease, osteogenesis imperfecta, cutaneous porphyria, orofacial cleft or episodic fever, were examined directly by WES between October 2018 and November 2021.

[Figure omitted. See PDF.]

Using a phenotype-based candidate gene approach, 169 index patients were screened by Sanger sequencing for variants in JAG1 (n = 22), ABCB11 & ATP8B1 (n = 94), and ABCB4 (n = 53). The remaining 17 index patients with diagnostic uncertainty were directly examined by WES. Nineteen of the 22 clinically diagnosed Alagille syndrome patients carried monoallelic P, LP or VUS variants in JAG1 (S3A Table). Forty-one of the 147 patients with non-syndromic cholestasis were homozygotes or compound heterozygotes for P/LP/VUS variants in either ABCB11 or ATP8B1 (32 patients with low-GGT cholestasis, S3B Table) or in ABCB4 (9 patients with high-GTT cholestasis, S3C Table). The 60 patients carrying monoallelic variants in JAG1 or biallelic variants in ABCB11, ATP8B1 or ABCB4 were excluded.

Histologic and immunohistologic study

Percutaneous liver-biopsy specimens were fixed in 4% paraformaldehyde and routinely processed for histological examination. Paraffin sections cut at 4 μm were stained with haematoxylin and eosin, Weigert-van Gieson with resorcin-fuchsin, periodic acid-Schiff reaction (PAS) with and without diastase pre-digestion, orcein, and Perls’ reaction. For immunohistochemical analysis, sections were incubated with anti-cytokeratin 7 antibody at a dilution of 1:100, anti-MDR3/ABCB4 rabbit polyclonal antibody at a dilution of 1:20, and anti-BSEP/ABCB11 rabbit polyclonal antibody at a dilution of 1:200 (details, S1 Table). Primary antibodies were detected by ultraView Universal DAB Detection Kit (Roche Tissue Diagnostics [formerly Ventana Medical Systems], Basel, Switzerland).

DNA isolation.

Genomic DNA was extracted from peripheral blood by QIAamp DNA Blood Kit (Qiagen, Hilden, Germany) with standard procedures.

Whole-exome sequencing

After mechanical fragmentation, exome libraries were constructed according to the manufacturer’s standard protocol with TruSeq® Exome Kit (Illumina, San Diego, CA). WES was accomplished using the Illumina NextSeq 500 platform. Alignment to the reference sequence (hg19 build) and variant calling was performed by Qiagen CLC Genomics Workbench 12 (https://digitalinsights.qiagen.com/). Identified variants were selected for annotation and interpretation using a 5% minor allele frequency (MAF) threshold taken from the gnomAD database. IGV Viewer [12] was used to display the data. Exome sequencing data are deposited in the General University Hospital in Prague and National Centre for Medical Genomics databases. Access conditioned by permission from the General University Hospital Ethics Committee.

Sanger sequencing

DNA sequencing of gel-purified PCR products (primer list, S2 Table) was performed by 3.1 Dye Terminator cycle sequencing kit and 3130 Genetic Analyzer electrophoresis (both Thermo Fisher Scientific, Waltham, MA).

Variant classification

Variants were classified according to American College of Medical Genetics and Genomics [ACMG] professional guidelines [13] using the VARSOME tool [14], published literature, variant databases NCBI ClinVar & Human Gene Mutation Database (HGMD), and population frequency information compiled in NCBI dbSNP. Sequence variations were assigned among the 5 categories benign, likely benign, variant of unknown significance (VUS), likely pathogenic (LP), or pathogenic (P). We characterized carriage of one P or LP variant as diagnostic for autosomal-dominant diseases and carriage of P/P, P/LP, or LP/LP variants as diagnostic for autosomal-recessive diseases.

Detection of copy number variations (CNV) and homozygosity regions

The command-line software toolkit CNVkit version 0.9.6CNV was used for CNV analysis of exome-sequencing data. Validation was performed by array comparative genome hybridization (CGH) using the commercially available oligonucleotide platforms GenetiSure Cyto 4x180K and SurePrint G3 CGH+SNP 4x180K (both Agilent Technologies, Santa Clara, CA). The latter platform was also used for detection of homozygosity regions. Data were analysed by CytoGenomics software version 5.1.2.1 (Agilent Technologies). UCSC Genome Browser, DECIPHER, NCBI ClinVar, OMIM, and DGV databases were used for assessment of clinical impacts of deletions and duplications.

Long range PCR and long-range amplicon single strand sequencing

A region containing the variants of interest in IFT172 was directly amplified from genomic DNA using TaKaRa LA Taq DNA Polymerase with 10x LA PCR Buffer II (TaKaRa, Mountain View, CA) with a two-step PCR protocol (primers, S2 Table). Initial denaturation at 94°C for 1min was followed by 30 cycles of 98°C for 10s denaturation and 64.1°C for 22min and 30s elongation. Final elongation was performed by incubation at 72°C for 10min. Product length was 22,800 bp. LR-PCR products were purified using SPRI magnetic beads (Beckman Coulter Life Sciences, Brea, CA) according to the manufacturer’s protocol, with 2μl used for Qubit 2.0 Fluorometric Quantitation (Beckman Coulter Life Sciences) dsDNA high sensitivity assay. Purified samples were sequenced by Pacific Biosciences Sequel system (PacBio, Menlo Park, CA) according to manufacturer’s protocol using SMRTbell Express Template Kit 2.0 and Sequel Sequencing Kit 3.0. To obtain highly accurate reads, Circular Consensus Sequence (CCS) analysis was performed using Pacific Biosciences SMRT Link (v6.0). CCS reads were aligned to human reference genome (hg19) using Minimap2 v. 2.24 [15], sorted with SAMtools and visualized in IGV browser.

Nasopharyngeal swab mRNA analysis

Total RNA was isolated from nasopharyngeal swabs using viRNAtrap and magnetic beads from GeneSpector (Prague, Czech Republic). RNA libraries were prepared using KAPA RNA HyperPrep Kit with RiboErase (Roche, Basel, Switzerland) according to manufacturer’s instructions. RNA sequencing (2x100 paired-end reads) was performed in 3 patients and family members using the NovaSeq 6000 system (Illumina, San Diego, CA) at the National Center for Medical Genomics in Prague. The resulting files in FASTQ format were subjected to quality control and trimmed using Atropos v.1.128 [16]. Gene-level abundances were estimated using Salmon v.1.3 [17] with Ensembl based annotation package EnsDb.Hsapiens.v75. Normalization and differential expression analyses were performed within the DESeq2 R package [18].

Coding DNA was transcribed from RNA isolated from nasopharyngeal swab using SuperScript™ IV Reverse Transcriptase (ThermoFisher), oligo dT 23VN and random hexamers following manufacturers protocol. A region encompassing F11R exons 1–10 was amplified using Phusion Hot Start Flex DNA Polymerase (Thermo Fisher Scientific) with a three step PCR protocol. The 1,052 bp product was purified using SPRI magnetic beads (Beckman Coulter Life Sciences, Brea, CA) and the region of interest was sequenced (primers, S2 Table).

Results

WES was performed in 51 clinically unrelated index subjects and in 58 available family members. Demographic characteristics and laboratory phenotype of the enrolled index subjects are presented in Table 1.

[Figure omitted. See PDF.]

In 26 index subjects (18 children at referral), initial Sanger sequencing had found no aetiologic variants in JAG1 (OMIM 601920), ABCB11 (OMIM 603201), ATP8B1 (OMIM 602397), or ABCB4 (OMIM 171060). In 8 single heterozygotes (5 children at referral) for P/LP variants in ABCB11, ATP8B1 (S4A and S4B Table) or ABCB4 (S4A and S4C Table), clinical findings (with disease) were discordant with heterozygosity for just one variant. WES did not reveal genetic causes of liver disease in these 34 patients; however, among the remaining 17 families (10 index patients were children at referral), unexpected molecular findings potentially relevant for genetic liver disease were obtained in 6 patients (Table 2).

[Figure omitted. See PDF.]

In 3 of these patients (ID F48CE680, F9CE723, and M18CE172), molecular diagnoses respectively of variegate porphyria (VP, OMIM #176200), polycystic kidney disease with or without polycystic liver disease 4 (PKD4, OMIM #263200), and nephronophthisis 11 (NPHP11, OMIM #613550, ref. [19]) were established. Two other index subjects harboured biallelic variants in IFT172 (OMIM 607386):

Patient 1 with biallelic variants in IFT172

A Roma man (ID M26RO684, Table 2) aged 26y, living with foster parents and without available birth-family members, had chronic liver disease with hepatomegaly since early childhood. Histopathologic study of a liver-biopsy specimen obtained at age 4y found preserved hepatic lobular and vascular architecture with well-glycogenated hepatocytes and trivial steatosis; fibrosis, inflammation, and accumulations of bile pigment were not seen (Fig 2).

[Figure omitted. See PDF.]

(A) Preserved lobular architecture with enlarged and pale hepatocytes (haematoxylin and eosin, original magnification 200x). (B) Interlobular bile duct deficiency with aberrant CK7 expression in some periportal hepatocytes (anti-CK7 immunostaining—CK7-expressing cells highlighted in brown / negative background blue, original magnification 400x). (C) Glycogen accumulation in hepatocytes with diffuse PAS reactivity, (D) lacking after diastase pre-digestion (original magnification 200x both). (E) Strong diffuse expression of ABCB11 on canalicular membranes of hepatocytes (anti-ABCB11 immunostaining, original magnification 400x). (F) Focally attenuated expression of ABCB4 on canalicular membranes of hepatocytes (anti-ABCB4 immunostaining, original magnification 400x).

Glycogen storage disease (GSD) was suspected and the patient was repeatedly examined by a specialist in genetic metabolic diseases. Activities of phosphorylase in leukocytes and of phosphorylase kinase and glycogen debrancher enzyme in erythrocytes investigated at age 9y were within normal ranges, leaving mild forms of GSD types I or III still under consideration. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were elevated (ALT 1.5x upper limit of normal [ULN], AST 2x ULN) without abnormal values for other biomarkers (serum bilirubin, GGT, blood glucose, lactate and lipid profile, renal-function indicators, and erythrocyte glycogen content) at age 11y. Dietary measures were introduced and metabolic-disease specialists supervised the patient till age 25y, when end-stage kidney disease of unknown aetiology requiring initiation of haemodialysis maintenance was diagnosed. Evaluation for renal transplant took place at the Institute of Clinical and Experimental Medicine.

On admission, apart from severely elevated urea and creatinine values, mildly increased serum AST activity (2.7x ULN) and severely increased cholestasis-biomarker enzyme activities (alkaline phosphatase 6.5x ULN, GGT 11.3x ULN) were observed, without hyperbilirubinemia. Ultrasonography revealed bilateral renal atrophy without abnormalities of liver or spleen. Percutaneous liver biopsy was repeated. The light-microscopy finding of mild hepatocellular glycogen deposition was unchanged. In addition, transfusional siderosis and lack of interlobular bile ducts in 5 of 14 portal tracts accompanied by mild features of chronic cholestasis were observed. Portal-tract fibrosis, hamartomatous or cystic bile-duct profiles, and hypoplasia of portal-vein radicles were absent; criteria for diagnosis of ductal plate malformation were not met. Canalicular expression of the homologues BSEP/ABCB11 and MDR3/ABCB4 was preserved, but for the latter was strikingly less prominent than for the former, suggesting ABCB4 disease. WES was requested to aid in diagnosis.

P/LP variants were detected among neither 66 neonatal/adult cholestasis genes [2] nor 21 genes mutated in GSD [20]. Bioinformatics analysis revealed a homozygous variant, rs780205001 c.167A>C (p.Lys56Thr), in IFT172, validated by Sanger sequencing (Fig 3A). The variant, rare in population databases (ExAC 0.0000119, GnomAD_exome 0.0000084, ALFA 0.005%) and not found in 110 unrelated Roma individuals, has not been identifiably reported in IFT172-related conditions. The lysine residue is highly conserved and a moderate physicochemical difference between lysine and threonine exists. The available evidence is currently insufficient to determine the role of this variant in disease. Therefore, the variant is classified as VUS in the NCBI ClinVar database (acc. no. VCV001015325.2). However, using the ACMG classification, Varsome suggested “warm” VUS (ACMG/AMP = PS4_supp (1p)+ PM2_supp (1p) + PM3_supp (0.5p)+ PP3 (1p)+ PP4 (1p)+ BP1 (-1p) = 3.5points >>VUS (tepid/warm)) and the prediction programmes PredictSNP (all MAPP, PhD-SNP, PolyPhen, SIFT and SNAP) and MutationTaster suggested likely pathogenicity. Accordingly, advanced modelling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) performed at Invitae (San Francisco, CA) indicates that this missense variant is expected to disrupt IFT172 protein function.

[Figure omitted. See PDF.]

Validation electropherograms of IFT172 partial genomic DNA sequence of exon 2 from index subjects M26RO684 (A) and M14CE762 (B), and exon 20 from index subject M14CE762 (C).

RNAseq of mRNA isolated from nasopharyngeal swabs confirmed that the variant NM_015662.3 r.257A>C was present in all reads from the IFT172 transcripts, without impact on IFT172 mRNA expression level, which was within population expression boundaries. No abnormal alternative splicing event in IFT172 was observed. The impact of the variant on IFT172 protein expression or subcellular localisation in the liver biopsy specimen could not be assessed due to the lack of a suitable antibody.

Parental unavailability precluded confirmation of homozygosity for the IFT172 variant by analysis of parental DNA samples. To distinguish between true homozygosity and hemizygosity for IFT172 c.167A>C, CNV analysis focused on the IFT172 locus ([GRCh37]: chr2: 27667244–27712610) was repeated. Read number did not fall. Moreover, WES data identified the variant as located in a 24.03Mb homozygosity region ([GRCh37], chr2:20440321–44471409). To validate this, we performed genomic hybridisation on two CGH arrays. In line with exomic data, no copy-number changes in the IFT172 region (2p23.3) or other CNV(s) explaining the proband’s phenotype were identified. The CGH SNP array detected loss of heterozygosity of autosomal regions (total range 322Mb; 11.2% of haploid autosomal genome). Coefficient of consanguinity (f) of the proband’s biological parents was 1/4. We also confirmed that IFT172 lay in a 27.2Mb homozygosity region (arr[GRCh37] 2p23.3(19824727_47021455)x2 hmz).

Within 1y the patient´s body mass index (BMI) recovered from 27.6 to 33 (obesity grade I). At age 26y the patient was listed for kidney transplantation, performed at age 27y.

The finding of a homozygous c.167A>C p.(Lys56Thr) “warm” VUS in IFT172 suggested short-rib thoracic dysplasia 10 (SRTD10, OMIM #615630) or Bardet-Biedl syndrome 20 (OMIM #619471). Further physical examination and roentgenograms of the chest, hips, legs, and feet revealed no bone malformations. Nor did ophthalmologic examination identify retinitis pigmentosa, typical in IFT172 deficiency (S1 Fig). Best corrected visual acuity was bilaterally normal (1.0 Snellen decimal values), as was intraocular pressure (19mmHg OD, 15mmHg OS). The patient interestingly had an abnormally thin retinal ganglion cell layer, documented using spectral domain optical coherence tomography (Spectralis, Heidelberg Engineering, Heidelberg, Germany).

Patient 2 with biallelic variants in IFT172

A caucasian boy (ID M14CE762, Table 2) born to unaffected parents, manifested severe icterus, hepatosplenomegaly, and coagulopathy aged 2mo, with hyperbilirubinemia (total bilirubin 18x ULN, direct bilirubin 27.3x ULN), abnormal hepatobiliary-injury biomarker values (AST 8.9x ULN, ALT 2.8x ULN), and hypercholanemia (30x ULN). GGT activity was within normal range. Light microscopy of a liver-biopsy specimen found giant-cell change of hepatocytes with cholestasis and fibrosis; steatosis and glycogen accumulation were absent. By age 10mo hyperbilirubinemia was nearly resolved; however, it has persisted (age 18y, total and direct bilirubin 1.4x and 1.1x ULN respectively) and findings on elastography (liver stiffness 9 kPa) indicate liver fibrosis and steatosis. The patient´s BMI is 20.6. Aside from nystagmus, physical-examination and laboratory findings are otherwise unremarkable, with in particular no skeletal malformations and no signs of kidney disease. He carries 3 variants in IFT172: On one allele he harbours the novel deletion c.2070del p.(Met690Ilefs*11, Fig 3C), classified as likely pathogenic (PVS1+PM2 = 9 points), and on the other (i.e., in trans) 2 single-nucleotide substitutions, rs776310391 c.157T>A p.(Phe53Ile) and rs746462745 c.164C>G p.(Thr55Ser) (Fig 3B), both classified as VUS according the ACMG guidelines and likely pathogenic based on the Association for Molecular Pathology criteria (c.157T>A: PM2_supp (1p) + PM3_supp (1p) +PP3 (1p) = 3 points >>VUS (tepid), c.164C>G: PM2_supp (1p) + PM3_supp (1p) + BP1 (-1p) + BP4 (-1p) = 0 points >>>VUS (ice cold)). Since both parents refused genetic testing, relative position of the variants was assessed by single strand sequencing of long-range PCR products encompassing all 3 mutated loci.

Carrier of biallelic variant in F11R

Finally, in a one-year-old (now 8y) Roma girl (ID F1RO453) with cholestatic cirrhosis, WES revealed a yet unreported homozygous splice-site variant, c.65-2A>T, in F11R (OMIM 605721), encoding the tight junction protein JAM1 (Figs 4 and S2).

[Figure omitted. See PDF.]

(A) Pedigree; arrow, index patient. (B) Sanger-sequencing electropherogram of F11R exon 2 amplified from the index patient´s gDNA. Arrow, homozygous F11R c.65-2A>T splice-site variant. (C) Agarose-gel electropherogram of PCR products encompassing exons 1–10 amplified from nasopharyngeal swabs F11R cDNA. (D) Sanger-sequencing electropherogram of the 983-bp fragment amplified from nasopharyngeal-swab cDNA of proband. Arrow, skipped exon 2. (E) Sanger-sequencing electropherogram of the 1052-bp fragment amplified from control nasopharyngeal-swab cDNA (wild-type sequence). Arrows, breakpoints. (F) Graphical representation of F11R exons 2, 3, and 4 coverage by RNAseq of nasopharyngeal-swab mRNA in IGV Viewer.

Both unaffected parents were asymptomatic heterozygotes for the F11R c.65-2A>T variant.

Since no liver-biopsy specimen suitable for mRNA analysis was available, we analyzed nasopharyngeal-swab mRNA by RNAseq. Apart from the expected in-frame deletion of 69 base pairs (NM_016946.6 r.144_212del) caused by skipping of exon 2 (confirmed by Sanger sequencing of F11R cDNA), RNAseq revealed no P/LP variants. The F11R variant is predicted to replace 24 amino-acid residues by serine (p.Cys22_Pro45delinsSer). Such an indel should impair the function of the altered protein, if expressed, by removing the motif Asn43-Asn44-Pro45, involved in trans-homophilic interaction of JAM1 cis-dimers [21]. Unfortunately, clinical pathogenicity of the variant is uncertain: The same homozygous variant exists in the proband´s sister, aged 7y and unaffected at this writing.

Extrahepatic diseases

Apart from the patients described above, other genetic diagnoses were suggested in another three probands (Table 3). These include osteogenesis imperfecta caused by a recently reported variant in COL1A2 (OMIM 120160) [22], familial periodic fever associated with a known variant in TNFRSF1A (OMIM 191190), and a combination of orofacial cleft with growth and mental retardation contributed by variants in TP63 (OMIM 603273), COL2A1 (OMIM 120140), and ZNF423 (OMIM 604557) [23].

[Figure omitted. See PDF.]

Discussion

In our cohort of 51 enrolled index patients, 34 of whom were extensively pre-screened by targeted gene approach, WES revealed unexpected findings in just 6 patients and other or additional genetic diagnoses in another 3 subjects. Multiple reasons for low efficiency can be adduced. Technical limitations of WES have been touched on; perhaps more important are difficulties with interpretation of variant pathogenicity. Sequencing of individual exomes provides lists of VUS never assessed experimentally. ACMG classification of individual variants is partially based on indirect indicators and in silico predictions–these in some situations may be misleading. That ClinVar classifications of numerous variants are equivocal, with several classifications of the same variant proposed, thus is unsurprising.

Concerning the capacity of WES to reveal rare unexpected principal and additional diagnoses, WES is obviously superior to sequencing a single gene or a disease gene panel. In the patient with jaundice associated with cutaneous porphyria (ID F48CE680), WES established the molecular diagnosis of variegate porphyria. This experience illustrates that diagnosis of disorders manifesting as cutaneous porphyria, with cholestasis and a negative family history but without a personal history of neurovisceral attacks, is scarcely possible without molecular-genetic studies.

Whereas congenital hepatic fibrosis was a clinical consideration in the second patient (ID F9CE723) despite the absence of cystic biliary-tract and renal disease detected after establishing the genetic diagnosis, ciliopathy gene variants were unexpected in the other three patients. In two, renal disease did not meet diagnostic criteria for nephronophthisis [19] before renal failure developed. Indeed, associations between liver disease and ciliopathy syndromes, including PKD4 and NPHP11, are published (review, Diamond et al. [24]).

The phenotype of IFT172 disease–short rib thoracic dysplasia 10 without polydactylv–varies greatly. It encompasses asphyxiating thoracic dysplasia (Jeune syndrome) [25], Mainzer-Saldino syndrome [25], isolated retinal degeneration [26], Bardet-Biedl syndrome [26, 27], and oral-facial-digital syndrome [28]. Some patients with Jeune or Meinzer-Saldino syndrome have nephronophthisis designated as NPHP17. Liver disease includes bile-duct abnormalities with cystic dilatation and periportal fibrosis. Although liver abnormalities may attract medical attention as the first sign of Jeune syndrome [29], they are considered minor and inconsistent ciliopathy features encountered in Jeune syndrome even with only mild skeletal malformations [30].

Non-syndromic cholestatic liver injury in IFT172 disease without skeletal malformations–and without bile-duct abnormalities–has never been identifiably reported. Our observation of 2 unrelated carriers of biallelic IFT172 variants with unexplained primary cholestatic liver disease, strengthened in the older patient (ID M26RO684) by adult-onset nephronophthisis, location of the homozygous IFT172 variant in a large homozygosity region inherited from consanguineous parents (cousins) and absence of candidate variants in the other 18 nephronophthisis genes, suggests association. Reduced retinal ganglion cell layer thickness observed in the older patient can be a sign of early pre-perimetric glaucoma [31]; however, given the age of the patient and normal intraocular pressure, a link with IFT172 cannot be excluded.

What diagnosis to assign to the sixth patient, who harbours a homozygous splice-site variant in F11R, remains unclear. F11R deficiency has never been identifiably reported in humans. Since F11r ^-/- mice are highly susceptible to liver injury [32–34] and since deficiency of other tight junction proteins, namely tight junction protein 2 and claudin-1, is associated with cholestatic liver disease [35, 36], to explain the proband´s phenotype as a consequence of JAM1 deficiency tempted us. That the proband´s unaffected sister carries the same F11R variant in homozygous state, however, casts doubt on the association. One might invoke incomplete penetrance driven by unknown genetic or environmental factors to explain this phenotypic variation, but studies in more families with JAM1 deficiency are essential to settle the matter.

An important favourable characteristic of genomic analysis technologies in general is that they are non-invasive. The clinical utility of biopsy in genetic liver diseases is limited [1]; in addition, patients or their parents may refuse liver biopsy, considering it unacceptably risky. Broader availability of RNAseq has opened the way to pathogenicity studies of candidate variants in liver-disease–causing genes expressed in extrahepatic epithelial tissues such as those accessible by nasopharyngeal swab (see S5 Table). As demonstrated in our patients with pathogenic variants in IFT172 and F11R, nasopharyngeal-swab mRNA analysis can be more informative than can immunohistologic assessment of expression of a limited panel of proteins in liver-biopsy specimens. Based on this experience, we believe that combined analysis of the whole exome and of nasopharyngeal-swab mRNA will make up for loss of some information hitherto gained in genetic liver diseases via histopathologic study.

In conclusion, we confirm the clinical utility of WES in patients with suspected genetic cholestasis. Combining WES with nasopharyngeal-swab mRNA analysis improves interpretation of WES data. Our findings suggest association of IFT172 variants with primary cholestatic liver disease which awaits confirmation by additional similar observations.

Supporting information

S1 File. List of referring centres.

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S1 Table. Antibodies.

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S2 Table. Sequence based reagents.

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S3 Table. Variants detected by initial Sanger sequencing in 60 excluded patients.

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S4 Table. Clinical diagnosis and variants found by initial Sanger sequencing in 34 enrolled patients.

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S5 Table. Nasopharyngeal swab expression of cholestasis and ciliopathy genes.

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S1 Fig. Retinal findings in patient homozygous for the IFT172 c.167A>C variant.

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S2 Fig. Molecular findings, family with JAM1 deficiency.

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S1 Checklist. STROBE statement—checklist of items that should be included in reports of observational studies.

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Acknowledgments

We thank Lucie Budišová for excellent technical assistance, Natálie Rysková for analysis of control Roma exomes and genomes, and all physicians who referred their patients for genetic testing.

Citation: Neřoldová M, Ciara E, Slatinská J, Fraňková S, Lišková P, Kotalová R, et al. (2023) Exome sequencing reveals IFT172 variants in patients with non-syndromic cholestatic liver disease. PLoS ONE 18(7): e0288907. https://doi.org/10.1371/journal.pone.0288907

About the Authors:

Magdaléna Neřoldová

Roles: Investigation, Validation, Visualization, Writing – original draft

Affiliations: Institute for Clinical and Experimental Medicine, Prague, Czech Republic, Institute of Medical Biochemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic

Elżbieta Ciara

Roles: Formal analysis, Investigation, Writing – review & editing

Affiliation: Department of Medical Genetics, The Children’s Memorial Health Institute, Warsaw, Poland

Janka Slatinská

Roles: Resources

Affiliation: Institute for Clinical and Experimental Medicine, Prague, Czech Republic

Soňa Fraňková

Roles: Resources

Affiliation: Institute for Clinical and Experimental Medicine, Prague, Czech Republic

ORICD: https://orcid.org/0000-0002-1462-5920

Petra Lišková

Roles: Investigation, Visualization, Writing – review & editing

Affiliations: Department of Ophthalmology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic, Department of Pediatrics and Inherited Metabolic Diseases, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic

Radana Kotalová

Roles: Resources

Affiliation: Department of Pediatrics, Second Faculty of Medicine, Charles University and Faculty Hospital Motol, Prague, Czech Republic

Janka Globinovská

Roles: Resources

Affiliation: Department of Pediatrics, Hospital Poprad, Poprad, Slovak Republic

Markéta Šafaříková

Roles: Data curation, Investigation

Affiliation: Institute of Medical Biochemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic

ORICD: https://orcid.org/0000-0003-2896-2108

Lucie Pfeiferová

Roles: Formal analysis

Affiliations: Institute of Medical Biochemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic, Department of Informatics and Chemistry, University of Chemistry and Technology in Prague, Prague, Czech Republic

Hana Zůnová

Roles: Data curation, Formal analysis, Investigation

Affiliation: Department of Biology and Medical Genetics, Second Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic

Lenka Mrázová

Roles: Data curation, Formal analysis, Investigation

Affiliation: Department of Pediatrics and Inherited Metabolic Diseases, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic

Viktor Stránecký

Roles: Formal analysis

Affiliation: Department of Pediatrics and Inherited Metabolic Diseases, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic

ORICD: https://orcid.org/0000-0002-2599-6479

Alena Vrbacká

Roles: Data curation, Investigation

Affiliation: Department of Pediatrics and Inherited Metabolic Diseases, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic

Ondřej Fabián

Roles: Investigation

Affiliations: Institute for Clinical and Experimental Medicine, Prague, Czech Republic, Department of Pathology and Molecular Medicine, 3rd Faculty of Medicine, Charles University and Thomayer Hospital, Prague, Czech Republic

Eva Sticová

Roles: Investigation

Affiliation: Institute for Clinical and Experimental Medicine, Prague, Czech Republic

ORICD: https://orcid.org/0000-0003-2486-6266

Daniela Skanderová

Roles: Resources

Affiliation: Department of Pathology, Faculty of Medicine and Dentistry, Palacky University Olomouc and Faculty Hospital, Olomouc, Czech Republic

Jan Šperl

Roles: Resources

Affiliation: Institute for Clinical and Experimental Medicine, Prague, Czech Republic

Marta Kalousová

Roles: Supervision, Writing – review & editing

Affiliation: Institute of Medical Biochemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic

Tomáš Zima

Roles: Funding acquisition, Supervision

Affiliation: Institute of Medical Biochemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic

ORICD: https://orcid.org/0000-0001-8518-6972

Milan Macek

Roles: Funding acquisition, Supervision

Affiliation: Department of Biology and Medical Genetics, Second Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic

Joanna Pawlowska

Roles: Resources

Affiliation: Department of Gastroenterology, Hepatology, Nutritional Disorders and Pediatrics, The Children’s Memorial Health Institute, Warsaw, Poland

A. S. Knisely

Roles: Investigation, Writing – review & editing

Affiliation: Diagnostik- und Forschungsinstitut für Pathologie, Medizinische Universität Graz, Graz, Austria

Stanislav Kmoch

Roles: Funding acquisition, Supervision, Writing – review & editing

Affiliation: Department of Pediatrics and Inherited Metabolic Diseases, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic

Milan Jirsa

Roles: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing

E-mail: [email protected]

Affiliations: Institute for Clinical and Experimental Medicine, Prague, Czech Republic, Institute of Medical Biochemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic

ORICD: https://orcid.org/0000-0001-7300-6735

References

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