Biallelic variants in NUDCD2 associated with a multiple malformation syndrome with cholestasis and renal failure

NudC‐like protein 2 (NUDCD2) is a 4‐exon protein‐coding gene at 5q34. The protein appears to act in concert with other genes regulating cell migration and microtubule extension. Early studies in model organisms show associations with LIS1, HERC2, and cohesin subunits via a co‐chaperone function with Heat shock protein 90 (Hsp90). It is a candidate gene for human pathology. We present two unrelated patients with biallelic variants in NUDCD2. Their phenotypes comprise similar dysmorphic facies, midline brain hypoplasia, hypothyroidism, pulmonary and aortic valve stenosis, severe dysfunction of the liver and kidneys, profound hypotonia, and early death. The cellular analysis demonstrates the absence of the NUDCD2 protein in fibroblasts of one patient with biallelic loss‐of‐function variants. The data suggest that NUDCD2 deficiency causes this recognizable syndrome that has features of a ciliopathy with additional complications.


| INTRODUCTION
Two unrelated patients with biallelic variants in candidate gene NUDCD2 present with similar structural and functional phenotypes.
Of the four identified variants, three are nonsense and one is missense. The patient with compound heterozygosity including the missense variant has no protein expression, implying that the missense variant also leads to loss of function. This suggests that NUDCD2 may be an autosomal recessive cause of the patients' phenotype. The shared phenotype includes features typically associated with ciliopathies and tubulinopathies. Thus far, limited studies of NUDCD2 indicate that it is a cochaperone active in creation and elongation of microtubules. This would be consistent with the shared phenotype.

| Case 1
The pregnancy was conceived via intrauterine insemination to a 32-yearold mother and 41-year-old father. This was their second pregnancy, the first having ended in spontaneous abortion at 18 weeks. The mother had a history of medullary thyroid cancer and multiple sclerosis, both in remission. She was on levothyroxine during pregnancy. Ultrasounds showed intrauterine growth restriction and the absence of the fetal left kidney. The family history was notable for three first trimester spontaneous abortions (out of five pregnancies) in the maternal grandmother, and bilateral congenital hearing loss in two distant paternal cousins.
Thorough evaluation revealed pulmonic valve stenosis, situs inversus totalis, absent left kidney, and right talipes equinovarus. She had dysmorphic features including non-synostotic scaphocephaly, low and posteriorly rotated ears, small mouth with high palate, an upward deviated tongue and profound hypotonia (Figure 1a).
Indirect hyperbilirubinemia resolved with phototherapy on days 2 and 3 of life with subsequent persistent direct hyperbilirubinemia of unclear etiology. Abdominal ultrasound showed normal gall bladder and no dilation of intrahepatic bile ducts. Within the first week of life, she was diagnosed with cholestasis and renal dysfunction progressing to chronic renal disease associated with acidosis. Newborn screening identified congenital hypothyroidism. Due to persistent and severe gastroesophageal reflux, dysphagia, and dysmotility, a gastrostomy tube was placed at 6 weeks of age. Over a short period of time, she developed hypertrophic cardiomyopathy of the left ventricle in addition to her known right ventricular outflow tract obstruction; severely complicating medical management.
Brain magnetic resonance imaging (MRI) identified a hypoplastic corpus callosum, brainstem, and cerebellar vermis. There was a globular appearance of the deep gray nuclei with poor visualization of the anterior limb internal capsule. Liver wedge biopsy showed paucity of interlobular bile ducts and hepatocellular and canalicular cholestasis with giant cell transformation but no significant portal fibrosis. A nasal brushing showed normal ciliary ultrastructure; ciliary motility was not assessed. She developed progressive acute-on-chronic hypoxemic respiratory failure and died at 13 weeks of age.
Autopsy confirmed the abnormalities of corpus callosum, brainstem, and cerebellar vermis. Both the pulmonary and aortic valves were small, the tricuspid and mitral leaflets were dysplastic, and the left ventricle showed concentric hypertrophy. There was exaggerated lobation and disorganized corticomedullary architecture of the remaining right kidney with unilocular cysts and focal ureteral stenosis. The uterus was unicornuate with the absence of the right horn and fallopian tube, and hypoplasia of the right ovary (Table 1).
Parental consent for research on autopsy tissue was obtained. NUDCD2 levels compared to fibroblast cell lines derived from healthy subjects ( Figure S1). This result reinforced the hypothesis that compound heterozygous variants in NUDCD2 (p.Glu125 Argfs*11 and p.M1?) led to the loss of NUDCD2 expression in this patient.

| Case 2
This was the first pregnancy to a 24-year-old mother and 27-year-old father. There was intrauterine growth restriction and labor was complicated by pre-eclampsia. The family history was notable for learning disabilities in the mother and breast cancer in the maternal grandmother. The father is reported to have had pancreatitis and bradycardia as a newborn, but neither as an ongoing concern.
The baby girl was delivered vaginally at 37 weeks. Birthweight was 2041 g (4th-10th centile) and birth length was 47 cm (10th-25th centile). She was noted after delivery to have a recessed chin, stridulous cry, and sacral dimple. She required a 10-day NICU stay because of neonatal pneumonia.
At 4 months of age, she was hospitalized due to failure to grow, nystagmus, and cholestasis of unknown etiology. The nystagmus and jerking head movements had started 6 weeks prior, raising concern for seizures. Brain MRI identified partial agenesis of the corpus callosum and hypoplastic pons. Electroencephalogram was normal. An echocardiogram identified mild pulmonary and aortic valve stenosis.
She was readmitted at 12 months of age with worsening cholestasis and newly diagnosed end-stage renal disease necessitating peritoneal dialysis. In the interim, she had been diagnosed with hypothyroidism.
A gastrostomy tube was placed because of persistent feeding difficulties and poor growth. Liver biopsy demonstrated a chronic cholestatic pattern of injury with cholangiopathy and periportal fibrosis. Kidney biopsy showed severe tubular atrophy and interstitial fibrosis and glomeruli with expanded Bowman's space. Dysmorphic features noted during admission included progressive microcephaly, bilateral esotropia, epicanthal folds, short nose, long philtrum, and low set ears T A B L E 1 Phenotypes of both cases. Highlighted features are shared between the two cases.   Yang et al. (2010) indicated that NUDCD2 stabilized the interaction between LIS1 and the chaperone Heatshock protein 90 (Hsp90) that is necessary for appropriate LIS1/dynein interaction. Subsequent work by the same group further defined that NUDCD2 is an Hsp90 co-chaperone that stabilizes the interaction between LIS1 and Hsp90  and that LIS1 (and thus the LIS1/Hsp90 interaction) promotes formation of activated dynein-1 (Htet et al., 2020).
NUDCD2, again as a co-chaperone of HSP90, additionally appears to stabilize myosin-9, which regulates actin dynamics (Chen et al., 2020). LIS1 and myosin-9 have opposite effects on cell migration. LIS1 promotes migration via microtubule formation. Myosin-9 inhibits migration by stabilizing the actin cytoskeleton. NUDCD2, in interacting with both of these processes, via HSP90, may be a major factor in cell migration and final localization (Chen et al., 2020).
Further studies at the cellular level suggest that NUDCD2 interacts with centrioles and microtubules in two other ways. It appears to regulate centriole duplication, avoiding over-production, via stabilization of HERC2 . It also promotes ciliogenesis by degrading the centriole cap, a process necessary for initiation of microtubule and thus primary (immotile) cilia formation (Liu et al., 2021).
Murine NUDCD2 has been identified. The protein is expressed in the early conceptus, embryonic ectoderm-both neural and non-neu-