DCDC2-Related Ciliopathy: Report of Six Polish Patients, Novel DCDC2 Variant, and Literature Review of Reported Cases

Introduction: The increasing usage of NGS technology has enabled the discovery of new causal genes in ciliopathies, including the DCDC2 gene. The aim of our study was to present the clinical, pathological and molecular report of six patients (from three unrelated families) with DCDC2 biallelic pathogenic variants. A detailed overview of the reported patients with DCDC2-related disease was provided. Material and methods: A retrospective chart review of the clinical, biochemical, pathological (liver histology) and molecular features of the study group was performed. The database PubMed (MEDLINE) was searched for relevant studies. Results: All the patients presented with cholestatic jaundice and elevated GGT; the mean age was 2 months. The initial liver biopsy was performed in four children at a mean age of 3 months (age range: 2–5 months). In all of them, features of cholestasis, portal fibrosis and mild portal inflammation were observed; in three of them ductular proliferation was observed. One patient had undergone liver transplantation (LTx) at 8 years of age. At hepatectomy, a biliary-pattern cirrhosis was observed. Only one patient presented with features of renal disease. Whole exome sequencing was performed in all patients at the last follow-up visit (mean age 10 years). Three different variants (one novel) in the DCDC2 gene were identified in the study group. With our six patients, a total of 34 patients with DCDC2-related hepatic ciliopathy were identified. The main clinical presentation of DCDC2-related ciliopathy was liver disease in the form of neonatal sclerosing cholangitis. The predominance of early and severe liver disease associated with no or mildly expressed kidney involvement was observed. Conclusions: Our findings expand the molecular spectrum of pathogenic DCDC2 variants, provide a more accurate picture of the phenotypic expression associated with molecular changes in this gene and confirm a loss of functional behaviour as the mechanism of disease.


Introduction
The term ciliopathy is attributed to the group of disorders resulting from abnormal formation or function of the primary (immotile) cilia [1,2]. As a result of its presence in nearly all tissues and organs, the impairment of their structure/function may result in heterogeneous phenotypes, ranging from single organ involvement to multi-systemic disorders [1,2]. However, some ciliopathies have variable tissue and organ predilection. In most of them, a combined kidney (fibrocystic renal disease) and liver (congenital hepatic fibrosis Diagnostics 2023, 13,1917 2 of 12 and/or Caroli disease) involvement is observed; this group is also called hepatorenal fibrocystic disorders (HRFCDs) [3]. Autosomal recessive polycystic kidney disease (ARPKD) is a prototype of HRFCD, and both along with autosomal dominant polycystic kidney disease (ADPKD) represent the most common ciliopathies [3,4]. In contrast to polycystic kidney disease, nephronophthisis-related ciliopathies (NPHP-RC), including isolated (#256100) and syndromic nephronopthisis (Senior-Loken syndrome, #266900; Joubert syndrome, #213300; Meckel Gruber syndrome, #249000) constitute the second group of HRFCDs [5].
The introduction of next-generation sequencing (NGS) technology, including targeted gene panels, whole-exome sequencing (WES) or even whole-genome sequencing (WGS) have now opened promising possibilities to identify the molecular background of genetic diseases [6,7]. An increasing usage of NGS technology has also enabled the discovery of new causal genes in ciliopathies, including the DCDC2 gene [8].
In 2015, Schueler et al. [8] provided the first report on two patients with nephronophthisis and early-onset severe hepatic fibrosis due to DCDC2 mutations. By immunofluorescence studies, the authors showed that DCDC2 localizes to the ciliary axoneme in the cilia of cholangiocytes and of renal epithelial cells [8]. The DCDC2 gene was then added to the list of genes causing renal-hepatic ciliopathy. In recent years, more and more patients with isolated liver disease or combined liver and kidney disease associated with DCDC2 pathogenic variants have been described. However, the detailed clinical presentation was not available in some of the reported cases, and thus created some confusion.
The aim of our study was to present the clinical, pathological and molecular report of six patients (from three unrelated families) with DCDC2 biallelic pathogenic variants. A detailed overview of the reported patients with DCDC2-related disease was provided.

Patients
The presentation at the time of diagnosis and detailed follow-up were described. Informed and written consent was obtained from the parents of patients. Ethical approval was obtained from the Children's Memorial Health Institute Bioethical Committee, Warsaw, Poland.
A retrospective chart review of patients' medical records concerning the biochemical (platelets, serum transaminases, total and direct serum bilirubin, gamma-glutamyl transpeptidase, internal normalized ratio, serum bile acids), pathological (microscopical examination of liver biopsy specimens) and molecular (targeted to cholestatic liver disease genes) were collected.

Liver Biopsy
The liver biopsy specimens, between 1.0 and 1.5 cm in length, were fixed in 4% formalin and stained by hematoxillin and eosin, the PAS method (periodic acid + Schiff reagent) and PAS method after diastase digestion, Azan method, reticulin impregnation and immunohistochemistry for cytokeratin detection was applied. To assess the histological activity of microscopical changes, the following categories of lesions were considered retrospectively: presence of inflammatory infiltrates in the portal spaces and lobules with or without piecemeal necrosis, degree of fibrosis, lobular disarray, rosette formations, proliferation of bile ducts and ductules with or without ductitis, lobular necrosis, hepatocyte giant cell transformation, steatosis and degenerative changes in the hepatocytes, canalicular bile plugs, cholestasis in the hepatocytes and in bile ducts, extramedullary hematopoesis.

Molecular Analysis
Genomic DNA was extracted from peripheral blood samples of the patients. Whole exome sequencing (WES) was performed in all the patients after the negative result of next-generation sequencing (NGS) of the targeted gene panel, created by The Children's Memorial Health Institute for the simultaneous sequencing of more than 1000 clinically relevant genes including 53 items related to cholestatic liver disorders. The nomenclature of molecular variants follows the Human Genome Variation Society guidelines (HGVS, http://varnomen.hgvs.org/) (accessed on 1 March 2023) using human cDNA sequencing of appropriate genes followed the Human Gene Mutation Database (HGMD, http://www.hgmd.cf.ac.uk) (accessed on 1 March 2023). American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) standardized guidelines for sequence-level variant classification in Mendelian diseases were used.

Literature Search
The database PubMed (MEDLINE) was searched for relevant studies on 10 March 2023. The following keyword-based strategy was used: ("neonatal sclerosing cholangitis" OR "NCS" AND ("DCDC2"). All studies, letters, and abstracts that contained sufficient data were included. Available data regarding the clinical presentation, biochemical and histological features, treatment, and outcome, as well as molecular characteristics, were extracted.

Patients' Presentation
Six Polish patients, including three males and three females, from three unrelated families were identified; see Table 1. The age at first clinical presentation was about 2 months (age range: 2nd day of life-3 months). All the patients presented with cholestatic jaundice and elevated GGT; in two (33%) of them, the presence of acholic stools in the neonatal period was observed.
The initial liver biopsy was performed in four children with a mean age of 3 months (age range: 2-5 months). In all of them, features of cholestasis, portal fibrosis and mild portal inflammation were observed; in three of them ductular proliferation was observed; see Table 2 and Figure 1. Two patients (Pt 1 and 6) were biopsied at later time points (3.5 and 2.5 years, respectively). Their specimens demonstrated a paucity of interlobular bile ducts and severe fibrosis.  One patient (Pt 1) had undergone liver transplantation (LTx) at 6 years of age. At hepatectomy, biliary-pattern cirrhosis was observed, see Table 2 and Figure 1.
Only one patient (Pt 1) of three patients who underwent magnetic resonance cholangiopancreatography (MRCP) presented with features of intrahepatic cholangiopathy; see Figure 2. The initial liver biopsy was performed in four children with a mean age of 3 months (age range: 2-5 months). In all of them, features of cholestasis, portal fibrosis and mild portal inflammation were observed; in three of them ductular proliferation was observed; see Table 2 and Figure 1. Two patients (Pt 1 and 6) were biopsied at later time points (3.5 and 2.5 years, respectively). Their specimens demonstrated a paucity of interlobular bile ducts and severe fibrosis.
One patient (Pt 1) had undergone liver transplantation (LTx) at 6 years of age. At hepatectomy, biliary-pattern cirrhosis was observed, see Table 2 and Figure 1.
Only one patient (Pt 1) of three patients who underwent magnetic resonance cholangiopancreatography (MRCP) presented with features of intrahepatic cholangiopathy; see Figure 2.   Only one patient (Pt 2) presented with features of renal disease; this patient wa diagnosed with right vesico-ureteral reflux and persistent urachus (Table 1).
Whole exome sequencing (WES) was performed in all patients at the last follow-u visit (2021-2022). The mean age at DCDC2-related ciliopathy diagnosis was 10 years (ag range 9 months-24 years).
Three different variants in the DCDC2 gene were identified in the study group (Table 1 All patients were compound heterozygotes, except one homozygote (Pt 6). Two sma deletions leading to frameshift and premature termination codon and one substitution acceptor splice site were detected. The deletion NM_016356.5: c.256de NP_057440.2:p.(Tyr86Thrfs*17) was not reported previously, but was detected in tran Only one patient (Pt 2) presented with features of renal disease; this patient was diagnosed with right vesico-ureteral reflux and persistent urachus (Table 1).
Whole exome sequencing (WES) was performed in all patients at the last follow-up visit (2021-2022). The mean age at DCDC2-related ciliopathy diagnosis was 10 years (age range 9 months-24 years).
Three different variants in the DCDC2 gene were identified in the study group (Table 1). All patients were compound heterozygotes, except one homozygote (Pt 6). Two small deletions leading to frameshift and premature termination codon and one substitution in acceptor splice site were detected. The deletion NM_016356.5: c.256del, NP_057440.2:p.(Tyr86Thrfs*17) was not reported previously, but was detected in trans with another known pathogenic variant (PM3). This variant was absent in null control chromosomes in GnomAD project (PM2_supporting). No computational evidence and in-silico tools support a deleterious effect, but this site is strongly conserved according to phyloP (score = 9.182) so is predicted a pathogenic outcome of this variant (PP3_supporting). It is expected, this frame shift (null) variant in gene DCDC2 cause loss of protein function due to NMD. Loss of function is a known mechanism of disease (PVS1). To date no clinical significance assessments have been submitted for this variant to ClinVar or HGMD database. According to ACMG/AMP classification variant received 12 points, which corresponds to the status: Pathogenic.
The most common presenting symptom was cholestatic jaundice and the mean age at presentation was 5 weeks (age range: 1 week-9 months). In most of them, an elevated GGT activity was reported. It is to be noted that a detailed clinical presentation was not available in some of the reported cases.
Seventeen patients (60%) required LTx at a mean age of 11 years old (range 8 months-25 years), while in two of them, a combined liver and kidney transplantation (LKTx) was performed.
Twenty-two patients had undergone liver biopsy, while in seven of them there were 2twobiopsies performed (including five histopathologic examinations of the explanted liver). In 13 patients, the liver biopsy was performed in the first 12 months of age (mean age: 5 months); in all of them features of cholestasis as well as fibrosis were observed.
Three patients (Pt 14, 21, 22) presented with psychomotor delay, while one of them had also microcephaly. One patient (Pt 16) presented with unilateral sensorineural hearing loss and brain imaging abnormalities. One patient (Pt 15) presented with internal carotid artery aneurysms.
Neonatal sclerosing cholangitis (NSC) is a rare and severe form of cholangiopathy. It was first reported in 1987 in eight children presenting in the neonatal period with cholestatic jaundice, hepatosplenomegaly, acholic stools, and elevated GGT, raising a clinical suspicion of biliary atresia (BA) [19]. Radiologic abnormalities of the intrahepatic and (in most of them) extrahepatic bile ducts, consistent with sclerosing cholangitis were observed. Jaundice subsided within several months of life; however, all the patients developed the clinical, biochemical and histological features of fibrosis or cirrhosis in the infancy [19].
In 2002 and subsequently 2014, French Authors described the first patients with neonatal cholestasis mimicking BA and a typical pattern of NSC in cholangiography [19,20]. Additionally, all the patients presented with diffuse ichthyosis. Molecular analyses identified CLDN1 (coding for claudin-1) defects, and the authors assigned a syndrome associating NSC and ichthyosis (#607626, NISCH syndrome) [20,21].
The first report of DCDC2 mutations (biallelic missense mutations or in-frame deletion) in NSC was provided by Girard et al. in a whole exome sequencing (WES) study in four patients from two unrelated consanguineous families [9]. Renal involvement was expressed heterogeneously, from its absence to congenital anomalies of the kidney and urinary tract (CAKUT) or development of renal insufficiency just after liver transplantation (LTx) [9]. In a study of Grammatikopoulos et al., 4 of 13 patients with NSC were found affected with mutations in DCDC2 [10]. None of them presented with renal disease. Since then, more and more patients with isolated liver disease (normal kidney function) or combined liver and kidney disease associated with DCDC2 pathogenic variants have been described. With our six patients, a total of 34 patients with DCDC2-related hepatic ciliopathy were identified.
Based on our case series and the literature review, we conclude that the main clinical presentation of DCDC2-related ciliopathy is liver disease in the form of NSC. Clinically the phenotype of NSC can mimic a biliary atresia, which is difficult to exclude, especially in the neonate presenting with cholestatic jaundice with elevated GGT and acholic stools. Besides cholestasis, the main characteristic feature of DCDC2 deficiency is biliary-tract inflammation with scarring. In the ultrastructural study in liver tissue, cholangiocyte injury was accompanied by the lack of identifiable primary cilia [10]. The Authors suggested that the absence of DCDC2 may be implicated either in the formation of "cytotoxic" bile or in dysregulation of the choanocytes' homeostatic mechanisms [10].
In patients with DCDC2-related cilopathy, renal phenotype was reported in a minority of them. However, based on the literature review, we would like to highlight that the absence of renal disorder at presentation does not exclude the possibility of renal involvement later in the patient's life. Thus, NSC patients should be regularly screened for renal disease. Two main phenotypes of renal involvement were observed, NPHP and congenital anomalies of the kidney and urinary tract (CAKUT) [8][9][10][11][12][13][14][15][16][17]. The characteristic phenomenon with predominance of early and severe liver disease associated with no or mildly expressed kidney involvement in DCDC2-related ciliopathy needs to be fully elucidated.
The clinical phenotype of two individuals (Pt 1, 2) carrying novel disease-causing DCDC2 variant was delineated, and collected clinical data were utilized to profile the disorder. In silico characterization of the newly identified frame shift deletion provided evidence that the consequence of this change on DCDC2 function is deleterious (12 points according ACMG classification). Loss of function is a known mechanism of DCDC2-related conditions (27 pathogenic LOF variants were reported in this gene). The variant affects first doublecortin functional domain-DCX1 (Figure 3). Our findings expand the molecular spectrum of pathogenic DCDC2 variants, provide a more accurate picture of the phenotypic expression associated with molecular changes in this gene and confirm a loss of functional behaviour as the mechanism of disease.
DCDC2 variant was delineated, and collected clinical data were utilized to profile the disorder. In silico characterization of the newly identified frame shift deletion provided evidence that the consequence of this change on DCDC2 function is deleterious (12 points according ACMG classification). Loss of function is a known mechanism of DCDC2-related conditions (27 pathogenic LOF variants were reported in this gene). The variant affects first doublecortin functional domain-DCX1 (Figure 3). Our findings expand the molecular spectrum of pathogenic DCDC2 variants, provide a more accurate picture of the phenotypic expression associated with molecular changes in this gene and confirm a loss of functional behaviour as the mechanism of disease.

Conclusions
1. The main clinical presentation of DCDC2-related ciliopathy is liver disease in the form of neonatal sclerosing cholangitis. 2. The predominance of early and severe liver disease associated with no or mildly expressed kidney involvement is observed in DCDC2-related ciliopathy. 3. Our findings expand the molecular spectrum of pathogenic DCDC2 variants, provide a more accurate picture of the phenotypic expression associated with molecular changes in this gene and confirm a loss of functional behaviour as the mechanism of disease.

1.
The main clinical presentation of DCDC2-related ciliopathy is liver disease in the form of neonatal sclerosing cholangitis.

2.
The predominance of early and severe liver disease associated with no or mildly expressed kidney involvement is observed in DCDC2-related ciliopathy.

3.
Our findings expand the molecular spectrum of pathogenic DCDC2 variants, provide a more accurate picture of the phenotypic expression associated with molecular changes in this gene and confirm a loss of functional behaviour as the mechanism of disease.

Institutional Review Board Statement:
The study has been approved by an ethics committee of The Children's Memorial Health Institute in Warsaw (institutional approved code 39/KB/2020).

Informed Consent Statement:
Informed consent was obtained from the parents of patients.
Data Availability Statement: All data generated or analysed during this study are included in this published article.

Conflicts of Interest:
All authors certify that they have no affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matter or materials discussed in this manuscript.