Biallelic known and novel DCDC2 variants in cholestatic liver disease: Phenotype–genotype observations in four children

Neonatal sclerosing cholangitis (NSC) is associated with progressing biliary fibrosis that often requires liver transplantation in childhood. Several recent studies have identified variants in DCDC2, encoding doublecortin domain‐containing protein 2 (DCDC2), expressed in primary cilia, that accompany syndromic disease and NSC. We report four patients with hepatobiliary disease associated with two novel homozygous or compound heterozygous variants in DCDC2. Three patients with protein‐truncating variants in DCDC2, expressing no DCDC2, presented with the originally described severe hepatic phenotype in infancy. One patient with a novel homozygous DCDC2 missense variant shows a markedly milder phenotype only manifest in childhood and with retained DCDC2 expression. Concomitant nephronophthisis is present in three patients and learning disability in two. This report widens the phenotypic spectrum of DCDC2‐associated hepatobiliary disease. Testing for DCDC2 expression and DCDC2 variants should be included in the evaluation of cholangiopathy of unknown aetiology in childhood as well as in infancy.

). Metabolic disorders were sought and not identified. Percutaneous liver biopsy did not yield a definitive diagnosis. Sonography showed normal gallbladder and kidneys.
Ursodeoxycholic acid therapy was initiated and cholestasis improved, but cirrhosis developed and was complicated by variceal bleeding. Aged 5 years, the patient is not obese and shows no signs of developmental delay or kidney disease. Audiometry findings are normal. She awaits LTX (Table S2).
Patient C is a boy, born after an uncomplicated full-term pregnancy, who presented aged 20 months with severe pronounced loss of body weight (8% over 4 months). The parents are consanguineous ( Figure 1), with no personal or familial history of liver disease.
Neonatal jaundice or pale stools were not reported. Growth and development had been normal. No malformations were identified.
Biomarker values suggested cholestatic hepatitis with elevated GGT, AST and ALT activities (Table S1). Metabolic disorders and infection were ruled out. Sonography found irregular liver texture, splenomegaly and normal gallbladder and kidneys. Percutaneous liver biopsy did not yield a definitive diagnosis. Nutritional support restored normal growth, with resolution of hyperbilirubinemia but persistence of biomarker value elevations (Table S1)

Key points
Variants in DCDC2 are associated with cholestatic liver disease often requiring liver transplantation in childhood.
Liver histology is characterized by ductal plate malformation (DPM). We report three patients in whom DCDC2 disease followed this pattern and a fourth with a milder disease, in whom symptoms appeared only later in childhood and DPM was not found. Thus, DCDC2-associated liver disease should also be considered in patients with onset of cholestasis after infancy and without DPM. stenosis of intrahepatic bile ducts without dilatation. The patient at present, aged 8 years, is clinically well. Renal function is still maintained (Table S1). He has no auditory defects (normal audiometry results) and is not obese or hypertensive.
Patient D, born after an uncomplicated full-term pregnancy, is the son of healthy non-consanguineous parents. He presented aged 1 month with jaundice and acholic stools. Growth and neurological development were normal. Malformations were not observed.
Metabolic diseases were excluded. Sonography found a small gallbladder. The common bile duct could not be identified. The kidneys were unremarkable. Intraoperative cholangiography aged 1.5 months showed a patent but hypoplastic-appearing intra-and extrahepatic biliary tract. Wedge liver biopsy did not yield a definitive diagnosis. Percutaneous liver biopsy aged 5 months found micronodular cirrhosis. Ascites developed and was managed by supportive care. The patient was clinically stable until age 12 years, when he developed acute encephalitis, with cerebral abscesses on imaging study. Although no organisms were identified, this responded to antibacterial treatment. Oesophageal variceal bleeding supervened and at age 13 years LTX was conducted. Renal function was noted as impaired during the episode of encephalitis, with sonographic evidence of fibrosis, and worsened after variceal haemorrhage, but recovered after LTX and was stable until age 15 years, when sonography indicated nephronophthisis. At age 18 years, renal transplantation was conducted with underlying chronic kidney disease stage 5. Aged 28 years, the patient is without intellectual or physical disability. He is under treatment for a B-cell lymphoproliferative disorder. Arterial hypertension has never been identified, hearing difficulties are not clinically manifest (audiometry has not been conducted), and he is not obese (Table S2).

| Histopathologic studies
The liver of patient A (3 months; Figure  Axonemal DCDC2 expression was not found; however, cholangiocyte cytoplasm marked diffusely. Both axonemal and cytoplasmic ACALT expression was seen. At hepatectomy (13 years; Figure 2D), cirrhosis had supervened. Principal bile ducts were largely lacking, with cholangiocyte disarray and basement-membrane sclerosis in those remaining. Axonemal DCDC2 expression again was not found (Table S2).

| DISCUSS ION
This study extends the genetic and phenotypic spectrum of DCDC2associated disease. Three of four patients had biallelic DCDC2  In summary, our study expands the palette of known DCDC2 variants and illustrates the value of complementing immunohistopathology in hepatobiliary disease with genetic investigation and vice versa. We recommend assessment for both DCDC2 protein expression and DCDC2 variants not only in NSC but also -as seen with patient C -in cholangiopathy of unknown aetiology manifesting in later life.

ACK N O WLE D G E M ENTS
We thank all patients and families for their cooperation and the Foundation, project number 501883972).

CO N FLI C T O F I NTE R E S T S TATE M E NT
None to declare.

E TH I C S S TATEM ENT
All tests performed in the patients were part of clinical assessment.
Patients gave written consent for publication of fully anonymised case reports. Additional ethical approval was not required.