BCL11A intellectual developmental disorder: defining the clinical spectrum and genotype-phenotype correlations.

An increasing number of individuals with intellectual developmental disorder (IDD) and heterozygous variants in BCL11A are identified, yet our knowledge of manifestations and mutational spectrum is lacking. To address this, we performed detailed analysis of 42 individuals with BCL11A-related IDD (BCL11A-IDD, a.k.a. Dias-Logan syndrome) ascertained through an international collaborative network, and reviewed 35 additional previously reported patients. Analysis of 77 affected individuals identified 60 unique disease-causing variants (30 frameshift, 7 missense, 6 splice-site, 17 stop-gain) and 8 unique BCL11A microdeletions. We define the most prevalent features of BCL11A-IDD: IDD, postnatal-onset microcephaly, hypotonia, behavioral abnormalities, autism spectrum disorder, and persistence of fetal hemoglobin (HbF), and identify autonomic dysregulation as new feature. BCL11A-IDD is distinguished from 2p16 microdeletion syndrome, which has a higher incidence of congenital anomalies. Our results underscore BCL11A as an important transcription factor in human hindbrain development, identifying a previously underrecognized phenotype of a small brainstem with a reduced pons/medulla ratio. Genotype-phenotype correlation revealed an isoform-dependent trend in severity of truncating variants: those affecting all isoforms are associated with higher frequency of hypotonia, and those affecting the long (BCL11A-L) and extra-long (-XL) isoforms, sparing the short (-S), are associated with higher frequency of postnatal microcephaly. With the largest international cohort to date, this study highlights persistence of fetal hemoglobin as a consistent biomarker and hindbrain abnormalities as a common feature. It contributes significantly to our understanding of BCL11A-IDD through an extensive unbiased multi-center assessment, providing valuable insights for diagnosis, management and counselling, and into BCL11A's role in brain development.

Structural variant calling and clinical interpretation in 6224 unsolved rare disease exomes.

Structural variants (SVs), including large deletions, duplications, inversions, translocations, and more complex events have the potential to disrupt gene function resulting in rare disease. Nevertheless, current pipelines and clinical decision support systems for exome sequencing (ES) tend to focus on small alterations such as single nucleotide variants (SNVs) and insertions-deletions shorter than 50 base pairs (indels). Additionally, detection and interpretation of large copy-number variants (CNVs) are frequently performed. However, detection of other types of SVs in ES data is hampered by the difficulty of identifying breakpoints in off-target (intergenic or intronic) regions, which makes robust identification of SVs challenging. In this paper, we demonstrate the utility of SV calling in ES resulting in a diagnostic yield of 0.4% (23 out of 5825 probands) for a large cohort of unsolved patients collected by the Solve-RD consortium. Remarkably, 8 out of 23 pathogenic SV were not found by comprehensive read-depth-based CNV analysis, resulting in a 0.13% increased diagnostic value.

Clustered de novo start-loss variants in GLUL result in a developmental and epileptic encephalopathy via stabilization of glutamine synthetase.

Glutamine synthetase (GS), encoded by GLUL, catalyzes the conversion of glutamate to glutamine. GS is pivotal for the generation of the neurotransmitters glutamate and gamma-aminobutyric acid and is the primary mechanism of ammonia detoxification in the brain. GS levels are regulated post-translationally by an N-terminal degron that enables the ubiquitin-mediated degradation of GS in a glutamine-induced manner. GS deficiency in humans is known to lead to neurological defects and death in infancy, yet how dysregulation of the degron-mediated control of GS levels might affect neurodevelopment is unknown. We ascertained nine individuals with severe developmental delay, seizures, and white matter abnormalities but normal plasma and cerebrospinal fluid biochemistry with de novo variants in GLUL. Seven out of nine were start-loss variants and two out of nine disrupted 5' UTR splicing resulting in splice exclusion of the initiation codon. Using transfection-based expression systems and mass spectrometry, these variants were shown to lead to translation initiation of GS from methionine 18, downstream of the N-terminal degron motif, resulting in a protein that is stable and enzymatically competent but insensitive to negative feedback by glutamine. Analysis of human single-cell transcriptomes demonstrated that GLUL is widely expressed in neuro- and glial-progenitor cells and mature astrocytes but not in post-mitotic neurons. One individual with a start-loss GLUL variant demonstrated periventricular nodular heterotopia, a neuronal migration disorder, yet overexpression of stabilized GS in mice using in utero electroporation demonstrated no migratory deficits. These findings underline the importance of tight regulation of glutamine metabolism during neurodevelopment in humans.

Bi-allelic variants in CELSR3 are implicated in central nervous system and urinary tract anomalies.

CELSR3 codes for a planar cell polarity protein. We describe twelve affected individuals from eleven independent families with bi-allelic variants in CELSR3. Affected individuals presented with an overlapping phenotypic spectrum comprising central nervous system (CNS) anomalies (7/12), combined CNS anomalies and congenital anomalies of the kidneys and urinary tract (CAKUT) (3/12) and CAKUT only (2/12). Computational simulation of the 3D protein structure suggests the position of the identified variants to be implicated in penetrance and phenotype expression. CELSR3 immunolocalization in human embryonic urinary tract and transient suppression and rescue experiments of Celsr3 in fluorescent zebrafish reporter lines further support an embryonic role of CELSR3 in CNS and urinary tract formation.

Pathogenic variants in SOX11 mimicking Pitt-Hopkins syndrome phenotype.

Pitt-Hopkins syndrome (PTHS) is a rare neurodevelopmental disorder characterised by severe intellectual disability (ID), distinctive facial features and autonomic nervous system dysfunction, caused by TCF4 haploinsufficiency. We clinically diagnosed with PTHS a 14 6/12 -year-old female, who had a normal status of TCF4. The pathogenic c.667del (p.Asp223MetfsTer45) variant in SOX11 was identified through whole exome sequencing (WES). SOX11 variants were initially reported to cause Coffin-Siris syndrome (CSS), characterised by growth restriction, moderate ID, coarse face, hypertrichosis and hypoplastic nails. However, recent studies have provided evidence that they give rise to a distinct neurodevelopmental disorder. To date, SOX11 variants are associated with a variable phenotype, which has been described to resemble CSS in some cases, but never PTHS. By reviewing both clinically and genetically 32 out of 82 subjects reported in the literature with SOX11 variants, for whom detailed information are provided, we found that 7/32 (22%) had a clinical presentation overlapping PTHS. Furthermore, we made a confirmation that overall SOX11 abnormalities feature a distinctive disorder characterised by severe ID, high incidence of microcephaly and low frequency of congenital malformations. Purpose of the present report is to enhance the role of clinical genetics in assessing the individual diagnosis after WES results.

Snijders Blok-Campeau Syndrome: Description of 20 Additional Individuals with Variants in CHD3 and Literature Review.

Snijders Blok-Campeau syndrome (SNIBCPS, OMIM# 618205) is an extremely infrequent disease with only approximately 60 cases reported so far. SNIBCPS belongs to the group of neurodevelopmental disorders (NDDs). Clinical features of patients with SNIBCPS include global developmental delay, intellectual disability, speech and language difficulties and behavioral disorders like autism spectrum disorder. In addition, patients with SNIBCPS exhibit typical dysmorphic features including macrocephaly, hypertelorism, sparse eyebrows, broad forehead, prominent nose and pointed chin. The severity of the neurological effects as well as the presence of other features is variable among subjects. SNIBCPS is caused likely by pathogenic and pathogenic variants in CHD3 (Chromodomain Helicase DNA Binding Protein 3), which seems to be involved in chromatin remodeling by deacetylating histones. Here, we report 20 additional patients with clinical features compatible with SNIBCPS from 17 unrelated families with confirmed likely pathogenic/pathogenic variants in CHD3. Patients were analyzed by whole exome sequencing and segregation studies were performed by Sanger sequencing. Patients in this study showed different pathogenic variants affecting several functional domains of the protein. Additionally, none of the variants described here were reported in control population databases, and most computational predictors suggest that they are deleterious. The most common clinical features of the whole cohort of patients are global developmental delay (98%) and speech disorder/delay (92%). Other frequent features (51-74%) include intellectual disability, hypotonia, hypertelorism, abnormality of vision, macrocephaly and prominent forehead, among others. This study expands the number of individuals with confirmed SNIBCPS due to pathogenic or likely pathogenic variants in CHD3. Furthermore, we add evidence of the importance of the application of massive parallel sequencing for NDD patients for whom the clinical diagnosis might be challenging and where deep phenotyping is extremely useful to accurately manage and follow up the patients.

De novo missense variants in phosphatidylinositol kinase PIP5KIγ underlie a neurodevelopmental syndrome associated with altered phosphoinositide signaling.

Phosphoinositides (PIs) are membrane phospholipids produced through the local activity of PI kinases and phosphatases that selectively add or remove phosphate groups from the inositol head group. PIs control membrane composition and play key roles in many cellular processes including actin dynamics, endosomal trafficking, autophagy, and nuclear functions. Mutations in phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] phosphatases cause a broad spectrum of neurodevelopmental disorders such as Lowe and Joubert syndromes and congenital muscular dystrophy with cataracts and intellectual disability, which are thus associated with increased levels of PI(4,5)P2. Here, we describe a neurodevelopmental disorder associated with an increase in the production of PI(4,5)P2 and with PI-signaling dysfunction. We identified three de novo heterozygous missense variants in PIP5K1C, which encodes an isoform of the phosphatidylinositol 4-phosphate 5-kinase (PIP5KIγ), in nine unrelated children exhibiting intellectual disability, developmental delay, acquired microcephaly, seizures, visual abnormalities, and dysmorphic features. We provide evidence that the PIP5K1C variants result in an increase of the endosomal PI(4,5)P2 pool, giving rise to ectopic recruitment of filamentous actin at early endosomes (EEs) that in turn causes dysfunction in EE trafficking. In addition, we generated an in vivo zebrafish model that recapitulates the disorder we describe with developmental defects affecting the forebrain, including the eyes, as well as craniofacial abnormalities, further demonstrating the pathogenic effect of the PIP5K1C variants.

Patients with DeSanto-Shinawi syndrome: Further extension of phenotype from Italy.

Here we describe three patients with neurodevelopmental disorders characterized by mild-to-moderate intellectual disability, mildly dysmorphic features, and hirsutism, all of which carry de novo sequence variants in the WW domain-containing adaptor of the coiled-coil (WAC) gene; two of these-c.167delA, p.(Asn56I1efs*136) and c.1746G>C, p.(Gln582His)-are novel pathogenic variants, and the third-c.1837C>T, p(Arg613*)-has been previously described. Diseases associated with WAC include DeSanto-Shinawi syndrome; to date, de novo heterozygous constitutional pathogenic WAC variants have caused a syndromic form of intellectual disability and mild dysmorphic features in 33 patients, yet potential associations with other clinical manifestations, such as oligomenorrhea and hyperandrogenism, remain unknown, because the phenotypic spectrum of the condition has not yet been delineated. The patient bearing the novel c.167delA WAC gene variant presented a normal psychomotor development, oligomenorrhea, hyperandrogenism, and hirsutism, and hirsutism was also observed in the patient with the c.1746G>C WAC gene variant. Hypertrichosis and hirsutism have been described in nine DeSanto-Shinawi patients, only in 17 of the 33 aforementioned patients thus far reported this aspect, and no hormonal-pattern data are available. In conclusion, we note that the pathogenic c.167delA WAC variant may be associated with a mild phenotype; and in addition to the neurodevelopmental problems nearly all DeSanto-Shinawi patients experience (i.e., intellectual disability and/or developmental delay), we recommend the addition of mild dysmorphic features, hirsutism, and hypertrichosis to this clinical presentation.

Proteome balance in ciliopathies: the OFD1 protein example.

The balance of protein synthesis and degradation is finely regulated and influences cellular homeostasis and biological processes (e.g., embryonic development and neuronal plasticity). Recent data demonstrated that centrosomal/ciliary proteins enable proteome control in response to spatial or microenvironmental stimuli. Here, we discuss recent discoveries regarding the role in the balance of the proteome of centrosomal/ciliary proteins associated with genetic disorders known as ciliopathies. In particular, OFD1 was the first example of a ciliopathy protein controlling both protein expression and autophagic/proteasomal degradation. Understanding the role of proteome balance in the pathogenesis of the clinical manifestations of ciliopathies may pave the way to the identification of a wide range of putative novel therapeutic targets for these conditions.

ATP6V0C variants impair V-ATPase function causing a neurodevelopmental disorder often associated with epilepsy.

The vacuolar H+-ATPase is an enzymatic complex that functions in an ATP-dependent manner to pump protons across membranes and acidify organelles, thereby creating the proton/pH gradient required for membrane trafficking by several different types of transporters. We describe heterozygous point variants in ATP6V0C, encoding the c-subunit in the membrane bound integral domain of the vacuolar H+-ATPase, in 27 patients with neurodevelopmental abnormalities with or without epilepsy. Corpus callosum hypoplasia and cardiac abnormalities were also present in some patients. In silico modelling suggested that the patient variants interfere with the interactions between the ATP6V0C and ATP6V0A subunits during ATP hydrolysis. Consistent with decreased vacuolar H+-ATPase activity, functional analyses conducted in Saccharomyces cerevisiae revealed reduced LysoSensor fluorescence and reduced growth in media containing varying concentrations of CaCl2. Knockdown of ATP6V0C in Drosophila resulted in increased duration of seizure-like behaviour, and the expression of selected patient variants in Caenorhabditis elegans led to reduced growth, motor dysfunction and reduced lifespan. In summary, this study establishes ATP6V0C as an important disease gene, describes the clinical features of the associated neurodevelopmental disorder and provides insight into disease mechanisms.

Crosstalk between cilia and autophagy: implication for human diseases.

Macroautophagy/autophagy is a self-degradative process necessary for cells to maintain their energy balance during development and in response to nutrient deprivation. Autophagic processes are tightly regulated and have been found to be dysfunctional in several pathologies. Increasing experimental evidence points to the existence of an interplay between autophagy and cilia. Cilia are microtubule-based organelles protruding from the cell surface of mammalian cells that perform a variety of motile and sensory functions and, when dysfunctional, result in disorders known as ciliopathies. Indeed, selective autophagic degradation of ciliary proteins has been shown to control ciliogenesis and, conversely, cilia have been reported to control autophagy. Moreover, a growing number of players such as lysosomal and mitochondrial proteins are emerging as actors of the cilia-autophagy interplay. However, some of the published data on the cilia-autophagy axis are contradictory and indicate that we are just starting to understand the underlying molecular mechanisms. In this review, the current knowledge about this axis and challenges are discussed, as well as the implication for ciliopathies and autophagy-associated disorders.

Targeting the MITF/APAF-1 axis as salvage therapy for MAPK inhibitors in resistant melanoma.

Melanoma is a deadly form of cancer characterized by remarkable therapy resistance. Analyzing the transcriptome of MAPK inhibitor sensitive- and resistant-melanoma, we discovered that APAF-1 is negatively regulated by MITF in resistant tumors. This study identifies the MITF/APAF-1 axis as a molecular driver of MAPK inhibitor resistance. A drug-repositioning screen identified quinacrine and methylbenzethonium as potent activators of apoptosis in a context that mimics drug resistance mediated by APAF-1 inactivation. The compounds showed anti-tumor activity in in vitro and in vivo models, linked to suppression of MITF function. Both drugs profoundly sensitize melanoma cells to MAPK inhibitors, regulating key signaling networks in melanoma, including the MITF/APAF-1 axis. Significant activity of the two compounds in inhibiting specific epigenetic modulators of MITF/APAF-1 expression, such as histone deacetylases, was observed. In summary, we demonstrate that targeting the MITF/APAF-1 axis may overcome resistance and could be exploited as a potential therapeutic approach to treat resistant melanoma.

SEMA6B variants cause intellectual disability and alter dendritic spine density and axon guidance.

Intellectual disability (ID) is a neurodevelopmental disorder frequently caused by monogenic defects. In this study, we collected 14 SEMA6B heterozygous variants in 16 unrelated patients referred for ID to different centers. Whereas, until now, SEMA6B variants have mainly been reported in patients with progressive myoclonic epilepsy, our study indicates that the clinical spectrum is wider and also includes non-syndromic ID without epilepsy or myoclonus. To assess the pathogenicity of these variants, selected mutated forms of Sema6b were overexpressed in Human Embryonic Kidney 293T (HEK293T) cells and in primary neuronal cultures. shRNAs targeting Sema6b were also used in neuronal cultures to measure the impact of the decreased Sema6b expression on morphogenesis and synaptogenesis. The overexpression of some variants leads to a subcellular mislocalization of SEMA6B protein in HEK293T cells and to a reduced spine density owing to loss of mature spines in neuronal cultures. Sema6b knockdown also impairs spine density and spine maturation. In addition, we conducted in vivo rescue experiments in chicken embryos with the selected mutated forms of Sema6b expressed in commissural neurons after knockdown of endogenous SEMA6B. We observed that expression of these variants in commissural neurons fails to rescue the normal axon pathway. In conclusion, identification of SEMA6B variants in patients presenting with an overlapping phenotype with ID and functional studies highlight the important role of SEMA6B in neuronal development, notably in spine formation and maturation and in axon guidance. This study adds SEMA6B to the list of ID-related genes.

De novo variants in ATP2B1 lead to neurodevelopmental delay.

Calcium (Ca2+) is a universal second messenger involved in synaptogenesis and cell survival; consequently, its regulation is important for neurons. ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) belongs to the family of ATP-driven calmodulin-dependent Ca2+ pumps that participate in the regulation of intracellular free Ca2+. Here, we clinically describe a cohort of 12 unrelated individuals with variants in ATP2B1 and an overlapping phenotype of mild to moderate global development delay. Additional common symptoms include autism, seizures, and distal limb abnormalities. Nine probands harbor missense variants, seven of which were in specific functional domains, and three individuals have nonsense variants. 3D structural protein modeling suggested that the variants have a destabilizing effect on the protein. We performed Ca2+ imaging after introducing all nine missense variants in transfected HEK293 cells and showed that all variants lead to a significant decrease in Ca2+ export capacity compared with the wild-type construct, thus proving their pathogenicity. Furthermore, we observed for the same variant set an incorrect intracellular localization of ATP2B1. The genetic findings and the overlapping phenotype of the probands as well as the functional analyses imply that de novo variants in ATP2B1 lead to a monogenic form of neurodevelopmental disorder.

Phenotypic spectrum of the recurrent TRPM3 p.(Val837Met) substitution in seven individuals with global developmental delay and hypotonia.

TRPM3 encodes a transient receptor potential cation channel of the melastatin family, expressed in the central nervous system and in peripheral sensory neurons of the dorsal root ganglia. The recurrent substitution in TRPM3: c.2509G>A, p.(Val837Met) has been associated with syndromic intellectual disability and seizures. In this report, we present the clinical and molecular features of seven previously unreported individuals, identified by exome sequencing, with the recurrent p.(Val837Met) variant and global developmental delay. Other shared clinical features included congenital hypotonia, dysmorphic facial features (broad forehead, deep-set eyes, and down turned mouth), exotropia, and musculoskeletal issues (hip dysplasia, hip dislocation, scoliosis). Seizures were observed in two of seven individuals (febrile seizure in one and generalized tonic-clonic seizures with atonic drops in another), and epileptiform activity was observed in an additional two individuals. This report extends the number of affected individuals to 16 who are heterozygous for the de novo recurrent substitution p.(Val837Met). In contrast with the initial report, epilepsy was not a mandatory feature observed in this series. TRPM3 pathogenic variation should be considered in individuals with global developmental delays, moderate-severe intellectual disability with, or without, childhood-onset epilepsy.

Bi-allelic variants in SPATA5L1 lead to intellectual disability, spastic-dystonic cerebral palsy, epilepsy, and hearing loss.

Spermatogenesis-associated 5 like 1 (SPATA5L1) represents an orphan gene encoding a protein of unknown function. We report 28 bi-allelic variants in SPATA5L1 associated with sensorineural hearing loss in 47 individuals from 28 (26 unrelated) families. In addition, 25/47 affected individuals (53%) presented with microcephaly, developmental delay/intellectual disability, cerebral palsy, and/or epilepsy. Modeling indicated damaging effect of variants on the protein, largely via destabilizing effects on protein domains. Brain imaging revealed diminished cerebral volume, thin corpus callosum, and periventricular leukomalacia, and quantitative volumetry demonstrated significantly diminished white matter volumes in several individuals. Immunofluorescent imaging in rat hippocampal neurons revealed localization of Spata5l1 in neuronal and glial cell nuclei and more prominent expression in neurons. In the rodent inner ear, Spata5l1 is expressed in the neurosensory hair cells and inner ear supporting cells. Transcriptomic analysis performed with fibroblasts from affected individuals was able to distinguish affected from controls by principal components. Analysis of differentially expressed genes and networks suggested a role for SPATA5L1 in cell surface adhesion receptor function, intracellular focal adhesions, and DNA replication and mitosis. Collectively, our results indicate that bi-allelic SPATA5L1 variants lead to a human disease characterized by sensorineural hearing loss (SNHL) with or without a nonprogressive mixed neurodevelopmental phenotype.

The role of OFD1 in selective autophagy.

Autophagy is a cellular self-degradative pathway. Our study unveiled a novel mechanism mediated by OFD1, the protein mutated in Oral-Facial-Digital type I syndrome, based on selective degradation of autophagic proteins, which enables cells to calibrate their self-degradation. We demonstrated that unrestrained autophagy contributes to renal cysts observed in Ofd1 mutants.

The TBC1D31/praja2 complex controls primary ciliogenesis through PKA-directed OFD1 ubiquitylation.

The primary cilium is a microtubule-based sensory organelle that dynamically links signalling pathways to cell differentiation, growth, and development. Genetic defects of primary cilia are responsible for genetic disorders known as ciliopathies. Orofacial digital type I syndrome (OFDI) is an X-linked congenital ciliopathy caused by mutations in the OFD1 gene and characterized by malformations of the face, oral cavity, digits and, in the majority of cases, polycystic kidney disease. OFD1 plays a key role in cilium biogenesis. However, the impact of signalling pathways and the role of the ubiquitin-proteasome system (UPS) in the control of OFD1 stability remain unknown. Here, we identify a novel complex assembled at centrosomes by TBC1D31, including the E3 ubiquitin ligase praja2, protein kinase A (PKA), and OFD1. We show that TBC1D31 is essential for ciliogenesis. Mechanistically, upon G-protein-coupled receptor (GPCR)-cAMP stimulation, PKA phosphorylates OFD1 at ser735, thus promoting OFD1 proteolysis through the praja2-UPS circuitry. This pathway is essential for ciliogenesis. In addition, a non-phosphorylatable OFD1 mutant dramatically affects cilium morphology and dynamics. Consistent with a role of the TBC1D31/praja2/OFD1 axis in ciliogenesis, alteration of this molecular network impairs ciliogenesis in vivo in Medaka fish, resulting in developmental defects. Our findings reveal a multifunctional transduction unit at the centrosome that links GPCR signalling to ubiquitylation and proteolysis of the ciliopathy protein OFD1, with important implications on cilium biology and development. Derangement of this control mechanism may underpin human genetic disorders.

A ZFYVE19 gene mutation associated with neonatal cholestasis and cilia dysfunction: case report with a novel pathogenic variant.

BACKGROUND: ZFYVE19 (Zinc Finger FYVE-Type Containing 19) mutations have most recently been associated to a novel type of high gamma-glutamyl transpeptidase (GGT), non-syndromic, neonatal-onset intrahepatic chronic cholestasis possibly associated to cilia dysfunction. Herein, we report a new case with further studies of whole exome sequencing (WES) and immunofluorescence in primary cilia of her cultured fibroblasts which confirm the observation. RESULTS: A now 5-year-old girl born to clinically healthy consanguineous Moroccan parents was assessed at 59 days of life due to severe cholestatic jaundice with increased serum bile acids and GGT, and preserved hepatocellular synthetic function. Despite fibrosis/cirrhosis and biliary ducts proliferation on liver biopsy suggested an extrahepatic biliary obstacle, normal intra-operatory cholangiography excluded biliary atresia. Under choleretic treatment, she maintained a clinically stable anicteric cholestasis but developped hyperlipidemia. After exclusion of the main causes of cholestasis by multiple tests, abnormal concentrations of sterols and WES led to a diagnosis of hereditary sitosterolemia (OMIM #618666), likely unrelated to her cholestasis. Further sequencing investigation revealed a homozygous non-sense mutation (p.Arg223Ter) in ZFYVE19 leading to a 222 aa truncated protein and present in both heterozygous parents. Immunofluorescence analysis of primary cilia on cultured skin fibroblasts showed a ciliary phenotype mainly defined by fragmented cilia and centrioles abnormalities. CONCLUSIONS: Our findings are consistent with and expands the recent evidence linking ZFYVE19 to a novel, likely non-syndromic, high GGT-PFIC phenotype with neonatal onset. Due to the possible role of ZFYVE19 in cilia function and the unprecedented coexistence of a coincidental hereditary sterol disorder in our case, continuous monitoring will be necessary to substantiate type of liver disease progression and/or possible emergence of a multisystemic involvement. What mentioned above confirms that the application of WES in children with undiagnosed cholestasis may lead to the identification of new causative genes, widening the knowledge on the pathophysiology.