Neonatal cholestasis (NC) affects 1 in 2500 term infants with varying etiologies (1). Ciliopathies constitute a group of genetic disorders associated with ciliary dysfunction leading to multiorgan involvement that can present with cholestasis (2). We report 2 siblings who presented with NC and paucity of bile ducts. They subsequently developed end-stage liver (ESLD) and renal disease (ESRD) with severe progressive cardiac dysfunction. A novel homozygous variant in the NEK8 (never in mitosis A-related kinase A) gene, associated with the multiorgan ciliopathy Nephronophthisis (NPHP) type 9, was identified in both siblings (3). INDEX PATIENT The patient (IV-3, Supplement Fig. 1A, Supplemental Digital Content, https://links.lww.com/MPG/B726) was born male, full-term, to healthy, consanguineous parents. He had worsening direct hyperbilirubinemia of 14 with elevated gamma-glutamyl transferase (GGT) up to 2306 units/L, within the first 2 weeks of life (Supplemental Table 1, Supplemental Digital Content, https://links.lww.com/MPG/B726). Infectious workup was nonrevealing. Abdominal ultrasound showed normal liver echogenicity without hepatic masses or intrahepatic ductal dilatation and 2 small kidneys. An intraoperative cholangiogram excluded biliary atresia (BA). The liver biopsy demonstrated paucity of interlobular bile ducts, cholangiolar proliferation, cholestasis, and mild bridging fibrosis (Fig. 1A and B). Genetic testing for Alagille syndrome including JAG1 deletion/duplication, progressive familial intrahepatic cholestasis (PFIC) and bile acid synthesis disorders (BASD) was negative. An echocardiogram showed trivial pulmonary stenosis. There was no family history of liver disease.FIGURE 1: (A) Trichrome stain of the wedge biopsy with bridging fibrosis at 2 months of age (Trichrome, 100×). (B) Index patient: cytokeratin 7 staining shows paucity of interlobular bile ducts with only bile ductules visible in the area shown, arrows (CK7, 200×). (C) Model of the protein structure illustrates the previously known mutations and the novel NEK8 variant detected in the protein kinase domain (starred). This illustration is adapted from Rajagopalan et al. Het = heterozygous; Hom = homozygous; RCC1 = Regulator of Chromosome Condensation 1; RHPD2 = renal-hepatic-pancreatic dysplasia 2.By 18 months, he progressed to ESLD with hepatopulmonary syndrome confirmed by contrast-enhanced echocardiogram. At 21 months, he received a deceased-donor whole orthotopic liver transplant (OLT). Despite normal liver function posttransplant, he developed progressive cardiac and renal disease. Subsequent echocardiograms revealed moderate aortic valve and mitral stenosis that culminated in an episode of cardiac arrest at 3 years age. This necessitated cardiac catheterization and aortic balloon valvuloplasty for worsening cardiac disease. His renal function declined requiring peritoneal dialysis at 3 years. Due to irreversibility of his cardiac condition and concomitant ESRD, heart and kidney transplant were not offered. He received palliative care and passed away at 5 years (Supplemental Table 1, Supplemental Digital Content, https://links.lww.com/MPG/B726). AFFECTED SIBLING Younger sister (IV-4) developed direct hyperbilirubinemia at birth. She underwent similar evaluations for infections, BA and other genetic disorders. Liver biopsy at 1 month of age demonstrated marked cholestasis, paucity of interlobular bile ducts, and mild cholangiolar proliferations, findings comparable with her older sibling (Supplemental Fig. 1B, Supplemental Digital Content, https://links.lww.com/MPG/B726). However, she had earlier manifestations of cardiac and renal involvement. By 22 months, she progressed to ESRD and ESLD, complicated by portal hypertension and severe malnutrition. Her echocardiogram showed moderate-to-severe aortic valve stenosis. She acutely decompensated and required mechanical ventilation and initiation of continuous venovenous hemofiltration. Eventually, she developed bradycardic arrest and expired at 30 months (Supplemental Table 1, Supplemental Digital Content, https://links.lww.com/MPG/B726). NEK8 GENE MUTATION This familial presentation raised concern for an inherited disorder. Whole exome gene sequencing (WES) identified a novel homozygous variant in the protein kinase domain of the NEK8 gene in both siblings (c. 35G>T, p. R12I). The parents were carriers of the same variant (Fig. 1C). Recessive or compound heterozygous variants in the NEK8 gene cause the ciliopathy NPHP9 (4). This NEK8 gene variant affects a highly conserved amino acid, which is conserved in Danio rerio (zebrafish) and the nematode Caenorhabditis elegans and is predicted to be damaging applying in-silico prediction tools (e.g. SIFT, Polyphen, and Mutation Taster). This variant was not identified in an ethnically matched cohort of 134 Hispanic controls using the data from the 1000 genome project, suggesting that this variant is likely pathogenic (5). DISCUSSION This report highlights 2 cases of homozygous, disease-causing mutations in NEK8 resulting ESLD leading to OLT in one and death in both associated with renal and cardiac dysfunctions. Both siblings presented with NC, elevated GGT, and paucity of bile ducts on liver biopsy. Workup for BA, PFIC, BASD, and Alagille syndrome was negative. Genetic testing demonstrated a novel homozygous variant in the NEK8 gene on chromosome 17 in both siblings, which encodes a kinase localized in centrosomes and cilia and is crucial for cell-cycle regulation (6). This variant is likely disease-causing as confirmed in a homozygous state in patients IV-3 and IV-4 and in a heterozygous state in both parents. Moreover, this variant is evolutionary conserved and predicted to be damaging. Finally, this variant has not been identified in over 100 ethnically matched control individuals (5). Otto et al (4) initially described the first human variants in NEK8 in 3 patients with no liver or heart disease. Frank et al (6) described a homozygous NEK8 variant in 3 fetuses out of 5 offsprings resulting in congenital heart disease, cystic dysplastic kidneys and pancreas, hypoplastic lungs, and hepatic fibrosis. Rajagopalan et al (7) similarly confirmed the multiorgan involvement caused by compound heterozygous NEK8 variants. All of these variants are localized in the regulator of chromosomal condensation (RCC1) domains (Fig. 1C). Grampa et al (8) identified additional variants in the RCC1 domains as well as the first variant in the kinase domain (Table 1 and Fig. 1C). Localization of NEK8 protein to cilia and centrosomes depends on the kinase activity and the noncatalytic RCC1 domains (3). NEK8 protein has multiple interaction partners involving different nephrocystins, which may also explain the extrarenal involvement of the heart and the liver. The mechanism of the bile duct paucity in NEK8 variants is not understood. However, NEK8 protein is involved in the YAP and HIPPO signaling pathways and its impairment can affect ciliogenesis and epithelialization (8).TABLE 1: Summary of clinical features of patients with NEK8 variantsOur patients’ variant is located in the N-terminal part of the NEK8 kinase domain whereas most NEK8 patients have variants affecting the RCC1 domains in the C-terminus of the gene (Fig. 1C). Unfortunately, the parents declined autopsies of the patients and genetic testing of the healthy siblings, which could have provided further insight for this condition. NEK8 variants may be underdiagnosed or under-reported because of miscarriages before diagnosis, or simply lack of testing. We suggest testing for this NEK8 variant when faced with NC of unknown etiology, particularly in the setting of ductal paucity on liver biopsy, elevated GGT and negative testing for Alagille syndrome and other elevated-GGT cholestasis entities, to provide a better prognosis and anticipate potential complications. It may also affect the decision for future transplantation.