Comparison of first-tier whole-exome sequencing with a multi-step traditional approach for diagnosing paediatric outpatients: An Italian prospective study.

BACKGROUND: The recent guidelines suggest the use of genome-wide analyses, such as whole exome sequencing (WES), at the beginning of the diagnostic approach for cases with suspected genetic conditions. However, in many realities it still provides for the execution of a multi-step pathway, thus requiring several genetic tests to end the so-called 'diagnostic odyssey'. METHODS: We reported the results of GENE Project (Genomic analysis Evaluation NEtwork): a multicentre prospective cohort study on 125 paediatric outpatients with a suspected genetic disease in which we performed first-tier trio-WES, including exome-based copy number variation analysis, in parallel to a 'traditional approach' of two/three sequential genetic tests. RESULTS: First-tier trio-WES detected a conclusive diagnosis in 41.6% of patients, way above what was found with routine genetic testing (25%), with a time-to-result of about 50 days. Notably, the study showed that 44% of WES-reached diagnoses would be missed with the traditional approach. The diagnostic rate (DR) of the two approaches varied in relation to the phenotypic class of referral and to the proportion of cases with a defined diagnostic suspect, proving the major difference for neurodevelopmental disorders. Moreover, trio-WES analysis detected variants in candidate genes of unknown significance (EPHA4, DTNA, SYNCRIP, NCOR1, TFDP1, SPRED3, EDA2R, PHF12, PPP1R12A, WDR91, CDC42BPG, CSNK1D, EIF3H, TMEM63B, RIPPLY3) in 19.4% of undiagnosed cases. CONCLUSION: Our findings represent real-practice evidence of how first-tier genome-wide sequencing tests significantly improve the DR for paediatric outpatients with a suspected underlying genetic aetiology, thereby allowing a time-saving setting of the correct management, follow-up and family planning.

Double somatic mosaicism in Cornelia de Lange syndrome.

Post-zygotic mosaicism is a well-known biological phenomenon characterized by the presence of genetically distinct lineages of cells in the same individual due to post-zygotic de novo mutational events. It has been identified in about 13% of Cornelia de Lange (CdLS) syndrome patients with a molecular diagnosis, an unusual high frequency. Here, we report the case of a patient affected by classic CdLS harboring post-zygotic mosaicism for two different likely pathogenic variants at the same nucleotide position in NIPBL. Double somatic mosaicism has never been reported in CdLS and only rarely recognized in human diseases. Possible pathogenetic mechanisms are discussed.

ARF1-related disorder: phenotypic and molecular spectrum.

PURPOSE: ARF1 was previously implicated in periventricular nodular heterotopia (PVNH) in only five individuals and systematic clinical characterisation was not available. The aim of this study is to provide a comprehensive description of the phenotypic and genotypic spectrum of ARF1-related neurodevelopmental disorder. METHODS: We collected detailed phenotypes of an international cohort of individuals (n=17) with ARF1 variants assembled through the GeneMatcher platform. Missense variants were structurally modelled, and the impact of several were functionally validated. RESULTS: De novo variants (10 missense, 1 frameshift, 1 splice altering resulting in 9 residues insertion) in ARF1 were identified among 17 unrelated individuals. Detailed phenotypes included intellectual disability (ID), microcephaly, seizures and PVNH. No specific facial characteristics were consistent across all cases, however microretrognathia was common. Various hearing and visual defects were recurrent, and interestingly, some inflammatory features were reported. MRI of the brain frequently showed abnormalities consistent with a neuronal migration disorder. CONCLUSION: We confirm the role of ARF1 in an autosomal dominant syndrome with a phenotypic spectrum including severe ID, microcephaly, seizures and PVNH due to impaired neuronal migration.

Rock around DYRK1A: Ethnic diversity, clinical challenges.

DYRK1A-related intellectual disability is a recently described syndrome characterized by microcephaly, global developmental delay, impaired speech development, and distinctive facial features, which let to define it as a recognizable syndrome. Here we report four new patients of different ethnicity, broadening the clinical phenotype of the condition and highlighting how ethnic influences in the facial appearance could make it less recognizable.

A clustering of heterozygous missense variants in the crucial chromatin modifier WDR5 defines a new neurodevelopmental disorder.

WDR5 is a broadly studied, highly conserved key protein involved in a wide array of biological functions. Among these functions, WDR5 is a part of several protein complexes that affect gene regulation via post-translational modification of histones. We collected data from 11 unrelated individuals with six different rare de novo germline missense variants in WDR5; one identical variant was found in five individuals and another variant in two individuals. All individuals had neurodevelopmental disorders including speech/language delays (n = 11), intellectual disability (n = 9), epilepsy (n = 7), and autism spectrum disorder (n = 4). Additional phenotypic features included abnormal growth parameters (n = 7), heart anomalies (n = 2), and hearing loss (n = 2). Three-dimensional protein structures indicate that all the residues affected by these variants are located at the surface of one side of the WDR5 protein. It is predicted that five out of the six amino acid substitutions disrupt interactions of WDR5 with RbBP5 and/or KMT2A/C, as part of the COMPASS (complex proteins associated with Set1) family complexes. Our experimental approaches in Drosophila melanogaster and human cell lines show normal protein expression, localization, and protein-protein interactions for all tested variants. These results, together with the clustering of variants in a specific region of WDR5 and the absence of truncating variants so far, suggest that dominant-negative or gain-of-function mechanisms might be at play. All in all, we define a neurodevelopmental disorder associated with missense variants in WDR5 and a broad range of features. This finding highlights the important role of genes encoding COMPASS family proteins in neurodevelopmental disorders.

Celiac disease prevalence and predisposing-HLA in a cohort of 93 Williams-Beuren syndrome patients.

Williams-Beuren syndrome is considered to be at increased risk for celiac disease, as for recent literature data and celiac disease guidelines, despite pathogenic mechanisms are still unclear. Our study analyzed the prevalence of autoimmune disorders, HLA DQ2 and/or DQ8 haplotypes, of transglutaminase antibodies and of diagnosis of celiac disease in a cohort of 93 Williams-Beuren syndrome's patients (mean age 21.26 years). Our study showed an increased prevalence of celiac disease equal to 10.8% (10/93 patients). We did not find a significant different frequency of predisposing HLA in subjects with Williams-Beuren syndrome compared to literature data in the general population (49.5% vs. 42.9%, with p > .1), nor a susceptibility to autoimmunity. This suggests that the increased prevalence of celiac disease in Williams-Beuren syndrome cannot be ascribed to HLA haplotype and may be related to other factors that still need to be identified in these patients.

Genotypes and phenotypes heterogeneity in PIK3CA-related overgrowth spectrum and overlapping conditions: 150 novel patients and systematic review of 1007 patients with PIK3CA pathogenetic variants.

BACKGROUND: Postzygotic activating PIK3CA variants cause several phenotypes within the PIK3CA-related overgrowth spectrum (PROS). Variant strength, mosaicism level, specific tissue involvement and overlapping disorders are responsible for disease heterogeneity. We explored these factors in 150 novel patients and in an expanded cohort of 1007 PIK3CA-mutated patients, analysing our new data with previous literature to give a comprehensive picture. METHODS: We performed ultradeep targeted next-generation sequencing (NGS) on DNA from skin biopsy, buccal swab or blood using a panel including phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin pathway genes and GNAQ, GNA11, RASA1 and TEK. Additionally, 914 patients previously reported were systematically reviewed. RESULTS: 93 of our 150 patients had PIK3CA pathogenetic variants. The merged PROS cohort showed that PIK3CA variants span thorough all gene domains, some were exclusively associated with specific PROS phenotypes: weakly activating variants were associated with central nervous system (CNS) involvement, and strongly activating variants with extra-CNS phenotypes. Among the 57 with a wild-type PIK3CA allele, 11 patients with overgrowth and vascular malformations overlapping PROS had variants in GNAQ, GNA11, RASA1 or TEK. CONCLUSION: We confirm that (1) molecular diagnostic yield increases when multiple tissues are tested and by enriching NGS panels with genes of overlapping 'vascular' phenotypes; (2) strongly activating PIK3CA variants are found in affected tissue, rarely in blood: conversely, weakly activating mutations more common in blood; (3) weakly activating variants correlate with CNS involvement, strong variants are more common in cases without; (4) patients with vascular malformations overlapping those of PROS can harbour variants in genes other than PIK3CA.

Atypical, Composite, or Blended Phenotypes: How Different Molecular Mechanisms Could Associate in Double-Diagnosed Patients.

In the last few years, trio-Whole Exome Sequencing (WES) analysis has revolutionized the diagnostic process for patients with rare genetic syndromes, demonstrating its potential even in non-specific clinical pictures and in atypical presentations of known diseases. Multiple disorders in a single patient have been estimated to occur in approximately 2-7.5% of diagnosed cases, with higher frequency in consanguineous families. Here, we report the clinical and molecular characterisation of eight illustrative patients for whom trio-WES allowed for identifing more than one genetic condition. Double homozygosity represented the causal mechanism in only half of them, whereas the other half showed peculiar multilocus combinations. The paper takes into consideration difficulties and learned lessons from our experience and therefore supports the powerful role of wide analyses for ascertaining multiple genetic diseases in complex patients, especially when a clinical suspicion could account for the majority of clinical signs. It finally makes clear how a patient's "deep phenotyping" might not be sufficient to suggest the presence of multiple genetic diagnoses but remains essential to validate an unexpected multilocus result from genetic tests.

Neuropsychiatric Functioning in CDLS: A Detailed Phenotype and Genotype Correlation.

Behavioural phenotype and autism-related traits of 38 patients affected by Cornelia de Lange syndrome (CdLS) were assessed using a specific neuropsychiatric protocol. Subsequently,we search for possible genotype-phenotype correlations comparing individuals with NIPBL variants and patients with negative molecular results. Firstly results showed a higher percentage of subjects with normal intellectual quotient (IQ) and borderline IQ; adaptive skills were lower than expected for age in all participants. 39.5% of the sample presented with autism spectrum disorder (ASD), NIPBL mutated individuals demonstrated a worse trend in comparison with the clinical diagnosis group. non-truncating individuals displayed no ASD and better communication abilities than truncating individuals. Findings increase our awareness of the strengths and weaknesses points in CdLS individuals.

Rare deleterious mutations of HNRNP genes result in shared neurodevelopmental disorders.

BACKGROUND: With the increasing number of genomic sequencing studies, hundreds of genes have been implicated in neurodevelopmental disorders (NDDs). The rate of gene discovery far outpaces our understanding of genotype-phenotype correlations, with clinical characterization remaining a bottleneck for understanding NDDs. Most disease-associated Mendelian genes are members of gene families, and we hypothesize that those with related molecular function share clinical presentations. METHODS: We tested our hypothesis by considering gene families that have multiple members with an enrichment of de novo variants among NDDs, as determined by previous meta-analyses. One of these gene families is the heterogeneous nuclear ribonucleoproteins (hnRNPs), which has 33 members, five of which have been recently identified as NDD genes (HNRNPK, HNRNPU, HNRNPH1, HNRNPH2, and HNRNPR) and two of which have significant enrichment in our previous meta-analysis of probands with NDDs (HNRNPU and SYNCRIP). Utilizing protein homology, mutation analyses, gene expression analyses, and phenotypic characterization, we provide evidence for variation in 12 HNRNP genes as candidates for NDDs. Seven are potentially novel while the remaining genes in the family likely do not significantly contribute to NDD risk. RESULTS: We report 119 new NDD cases (64 de novo variants) through sequencing and international collaborations and combined with published clinical case reports. We consider 235 cases with gene-disruptive single-nucleotide variants or indels and 15 cases with small copy number variants. Three hnRNP-encoding genes reach nominal or exome-wide significance for de novo variant enrichment, while nine are candidates for pathogenic mutations. Comparison of HNRNP gene expression shows a pattern consistent with a role in cerebral cortical development with enriched expression among radial glial progenitors. Clinical assessment of probands (n = 188-221) expands the phenotypes associated with HNRNP rare variants, and phenotypes associated with variation in the HNRNP genes distinguishes them as a subgroup of NDDs. CONCLUSIONS: Overall, our novel approach of exploiting gene families in NDDs identifies new HNRNP-related disorders, expands the phenotypes of known HNRNP-related disorders, strongly implicates disruption of the hnRNPs as a whole in NDDs, and supports that NDD subtypes likely have shared molecular pathogenesis. To date, this is the first study to identify novel genetic disorders based on the presence of disorders in related genes. We also perform the first phenotypic analyses focusing on related genes. Finally, we show that radial glial expression of these genes is likely critical during neurodevelopment. This is important for diagnostics, as well as developing strategies to best study these genes for the development of therapeutics.

Lithium as a possible therapeutic strategy for Cornelia de Lange syndrome.

Cornelia de Lange Syndrome (CdLS) is a rare developmental disorder affecting a multitude of organs including the central nervous system, inducing a variable neurodevelopmental delay. CdLS malformations derive from the deregulation of developmental pathways, inclusive of the canonical WNT pathway. We have evaluated MRI anomalies and behavioral and neurological clinical manifestations in CdLS patients. Importantly, we observed in our cohort a significant association between behavioral disturbance and structural abnormalities in brain structures of hindbrain embryonic origin. Considering the cumulative evidence on the cohesin-WNT-hindbrain shaping cascade, we have explored possible ameliorative effects of chemical activation of the canonical WNT pathway with lithium chloride in different models: (I) Drosophila melanogaster CdLS model showing a significant rescue of mushroom bodies morphology in the adult flies; (II) mouse neural stem cells restoring physiological levels in proliferation rate and differentiation capabilities toward the neuronal lineage; (III) lymphoblastoid cell lines from CdLS patients and healthy donors restoring cellular proliferation rate and inducing the expression of CyclinD1. This work supports a role for WNT-pathway regulation of CdLS brain and behavioral abnormalities and a consistent phenotype rescue by lithium in experimental models.

Not Only Diagnostic Yield: Whole-Exome Sequencing in Infantile Cardiomyopathies Impacts on Clinical and Family Management.

Whole-exome sequencing (WES) is a powerful and comprehensive tool for the genetic diagnosis of rare diseases, but few reports describe its timely application and clinical impact on infantile cardiomyopathies (CM). We conducted a retrospective analysis of patients with infantile CMs who had trio (proband and parents)-WES to determine whether results contributed to clinical management in urgent and non-urgent settings. Twenty-nine out of 42 enrolled patients (69.0%) received a definitive molecular diagnosis. The mean time-to-diagnosis was 9.7 days in urgent settings, and 17 out of 24 patients (70.8%) obtained an etiological classification. In non-urgent settings, the mean time-to-diagnosis was 225 days, and 12 out of 18 patients (66.7%) had a molecular diagnosis. In 37 out of 42 patients (88.1%), the genetic findings contributed to clinical management, including heart transplantation, palliative care, or medical treatment, independent of the patient's critical condition. All 29 patients and families with a definitive diagnosis received specific counseling about recurrence risk, and in seven (24.1%) cases, the result facilitated diagnosis in parents or siblings. In conclusion, genetic diagnosis significantly contributes to patients' clinical and family management, and trio-WES should be performed promptly to be an essential part of care in infantile cardiomyopathy, maximizing its clinical utility.

Defining the genotypic and phenotypic spectrum of X-linked MSL3-related disorder.

PURPOSE: We sought to delineate the genotypic and phenotypic spectrum of female and male individuals with X-linked, MSL3-related disorder (Basilicata-Akhtar syndrome). METHODS: Twenty-five individuals (15 males, 10 females) with causative variants in MSL3 were ascertained through exome or genome sequencing at ten different sequencing centers. RESULTS: We identified multiple variant types in MSL3 (ten nonsense, six frameshift, four splice site, three missense, one in-frame-deletion, one multi-exon deletion), most proven to be de novo, and clustering in the terminal eight exons suggesting that truncating variants in the first five exons might be compensated by an alternative MSL3 transcript. Three-dimensional modeling of missense and splice variants indicated that these have a deleterious effect. The main clinical findings comprised developmental delay and intellectual disability ranging from mild to severe. Autism spectrum disorder, muscle tone abnormalities, and macrocephaly were common as well as hearing impairment and gastrointestinal problems. Hypoplasia of the cerebellar vermis emerged as a consistent magnetic resonance image (MRI) finding. Females and males were equally affected. Using facial analysis technology, a recognizable facial gestalt was determined. CONCLUSION: Our aggregated data illustrate the genotypic and phenotypic spectrum of X-linked, MSL3-related disorder (Basilicata-Akhtar syndrome). Our cohort improves the understanding of disease related morbidity and allows us to propose detailed surveillance guidelines for affected individuals.

Complex nutritional deficiencies in a large cohort of Italian patients with Cornelia de Lange syndrome spectrum.

Cornelia de Lange syndrome Spectrum (CdLSp) is characterized by intellectual disability, facial dysmorphisms, and growth impairment. Although eating difficulties are a well-known feature of the disease, there is no data regarding the nutritional deficiencies of these patients. The food intake was tracked using a dietary transcription provided by the family/caregivers, biochemical nutritional parameters were measured with laboratory tests and through an accurate clinical evaluation of the incidence of qualitative and quantitative imbalances in a cohort of 73 patients with CdLSp ware determined. Of these 73, 62 (85%) subjects provided a complete and detailed dietary transcription. In the studied population, a quantitative caloric imbalance in 47/62 (76%) subjects was observed. The caloric intake was low in 27/62 (43%) subjects whereas excessive in 20/62 (33%). Only 15/62 (24%) had an optimum caloric intake. Regarding micronutrients, a calcium intake deficiency in 32% of the patients (20/62) was observed. Blood tests revealed a low iron level in 22/73 (30%) of the patients and 25(OH)D deficiency in 49/73 (67%). Serum hypocalcemia was not evidenced. Qualitative and quantitative imbalances resulted in more frequent than expected in CdLSp patients. A qualitative imbalance was more prevalent in younger patients while in older patients prevailed mainly a quantitative disproportion. We found no statistically meaningful correlation between dietary imbalances, genetic, or clinical parameters. Our findings highlight the need for further studies to evaluate the basal metabolic rate of CdLSp patients and find a correlation with their growth impairment.

Nissen fundoplication in Cornelia de Lange syndrome spectrum: Who are the potential candidates?

Cornelia de Lange spectrum (CdLSp) is a rare genetic condition characterized by intellectual disability, facial dysmorphisms, major malformations, growth impairment, and development delay. Approximately 80% of CdLSp patients have gastroesophageal reflux disease (GERD) with a varied clinical presentation. The aim of this study is to define potential clinical/genetic risk factors based on the clinical phenotype description of CdLSp patients with severe GERD who underwent surgical treatment. We retrospectively collected data from 23 CdLSp patients, 13 females and 10 males. Mean age of the patients undergoing surgical treatment was of 4 years. 21/23 (91%) had a molecular characterization, of which 21/21 (100%) had a NIPBL gene mutation, while 2/23 (9%) did not have a genetical characterization, only a clinical diagnosis. Most of our patients presented a moderate-severe severity score (21/23, 91%) with limb malformations evidenced in 10/23 (44%) of our patients and a moderate-severe intellectual disability in 20/23 (87%). Therefore, CdLSp patients harboring NIPBL variants, upper limb malformations and severe psychomotor delay are more likely to suffer from severe GERD, not responsive to proton pump inhibitors treatment. These features should be considered as clinical markers for potentially severe GERD that might require surgical treatment.

Phenotypic spectrum and transcriptomic profile associated with germline variants in TRAF7.

PURPOSE: Somatic variants in tumor necrosis factor receptor-associated factor 7 (TRAF7) cause meningioma, while germline variants have recently been identified in seven patients with developmental delay and cardiac, facial, and digital anomalies. We aimed to define the clinical and mutational spectrum associated with TRAF7 germline variants in a large series of patients, and to determine the molecular effects of the variants through transcriptomic analysis of patient fibroblasts. METHODS: We performed exome, targeted capture, and Sanger sequencing of patients with undiagnosed developmental disorders, in multiple independent diagnostic or research centers. Phenotypic and mutational comparisons were facilitated through data exchange platforms. Whole-transcriptome sequencing was performed on RNA from patient- and control-derived fibroblasts. RESULTS: We identified heterozygous missense variants in TRAF7 as the cause of a developmental delay-malformation syndrome in 45 patients. Major features include a recognizable facial gestalt (characterized in particular by blepharophimosis), short neck, pectus carinatum, digital deviations, and patent ductus arteriosus. Almost all variants occur in the WD40 repeats and most are recurrent. Several differentially expressed genes were identified in patient fibroblasts. CONCLUSION: We provide the first large-scale analysis of the clinical and mutational spectrum associated with the TRAF7 developmental syndrome, and we shed light on its molecular etiology through transcriptome studies.

Double homozygosity in CEP57 and DYNC2H1 genes detected by WES: Composite or expanded phenotype?

BACKGROUND: In the last few years trio-whole exome sequencing (WES) analysis has demonstrated its potential in obtaining genetic diagnoses even in nonspecific clinical pictures and in atypical presentations of known diseases. Moreover WES allows the detection of variants in multiple genes causing different genetic conditions in a single patient, in about 5% of cases. The resulting phenotype may be clinically discerned as variability in the expression of a known phenotype, or as a new unreported syndromic condition. METHODS: Trio-WES was performed on a 4-month-old baby with a complex clinical presentation characterized by skeletal anomalies, congenital heart malformation, congenital hypothyroidism, generalized venous and arterial hypoplasia, and recurrent infections. RESULTS: WES detected two different homozygous variants, one in CEP57, the gene responsible for mosaic variegated aneuploidy syndrome 2, the other in DYNC2H1, the main gene associated with short-rib thoracic dysplasia. CONCLUSION: The contribution of these two different genetic causes in determining the phenotype of our patient is discussed, including some clinical signs not explained by the detected variants. The report then highlights the role of WES in providing complete and fast diagnosis in patients with complex presentations of rare genetic syndromes, with important implications in the assessment of recurrence risk.

A novel nonsense and inactivating variant of ST3GAL3 in two infant siblings suffering severe epilepsy and expressing circulating CA19.9.

Three missense variants of ST3GAL3 are known to be responsible for a congenital disorder of glycosylation determining a neurodevelopmental disorder (intellectual disability/epileptic encephalopathy). Here we report a novel nonsense variant, p.Y220*, in two dichorionic infant twins presenting a picture of epileptic encephalopathy with impaired neuromotor development. Upon expression in HEK-293T cells, the variant appears totally devoid of enzymatic activity in vitro, apparently accumulated with respect to the wild-type or the missense variants, as detected by western blot, and in large part properly localized in the Golgi apparatus, as assessed by confocal microscopy. Both patients were found to efficiently express the CA19.9 antigen in the serum despite the total loss of ST3GAL3 activity, which thus appears replaceable from other ST3GALs in the synthesis of the sialyl-Lewis a epitope. Kinetic studies of ST3GAL3 revealed a strong preference for lactotetraosylceramide as acceptor and gangliotetraosylceramide was also efficiently utilized in vitro. Moreover, the p.A13D missense variant, the one maintaining residual sialyltransferase activity, was found to have much lower affinity for all suitable substrates than the wild-type enzyme with an overall catalytic efficiency almost negligible. Altogether the present data suggest that the apparent redundancy of ST3GALs deduced from knock-out mouse models only partially exists in humans. In fact, our patients lacking ST3GAL3 activity synthesize the CA19.9 epitope sialyl-Lewis a, but not all glycans necessary for fine brain functions, where the role of minor gangliosides deserves further attention.

Cohesin complex-associated holoprosencephaly.

Marked by incomplete division of the embryonic forebrain, holoprosencephaly is one of the most common human developmental disorders. Despite decades of phenotype-driven research, 80-90% of aneuploidy-negative holoprosencephaly individuals with a probable genetic aetiology do not have a genetic diagnosis. Here we report holoprosencephaly associated with variants in the two X-linked cohesin complex genes, STAG2 and SMC1A, with loss-of-function variants in 10 individuals and a missense variant in one. Additionally, we report four individuals with variants in the cohesin complex genes that are not X-linked, SMC3 and RAD21. Using whole mount in situ hybridization, we show that STAG2 and SMC1A are expressed in the prosencephalic neural folds during primary neurulation in the mouse, consistent with forebrain morphogenesis and holoprosencephaly pathogenesis. Finally, we found that shRNA knockdown of STAG2 and SMC1A causes aberrant expression of HPE-associated genes ZIC2, GLI2, SMAD3 and FGFR1 in human neural stem cells. These findings show the cohesin complex as an important regulator of median forebrain development and X-linked inheritance patterns in holoprosencephaly.

Diagnostic Yield of an Algorithm for Neonatal and Infantile Cholestasis Integrating Next-Generation Sequencing.

OBJECTIVE: To evaluate the performance of a diagnostic protocol for neonatal/infantile cholestasis in which the main clinical patterns steered the early use of different genetic testing strategies. STUDY DESIGN: An observational study was conducted between 2012 and 2017 in a tertiary care setting on a prospective cohort of children with cholestasis occurring at ≤1 year of age and persisting ≥6 weeks, to measure the detection rate of underlying monogenic diseases. After the exclusion of biliary atresia, a clinically driven genetic testing was performed, entailing 3 different approaches with different wideness: confirmatory single-gene testing; focused virtual panels; and wide search through trio whole-exome sequencing. RESULTS: We enrolled 125 children (66 female, median age 2 months); 96 (77%) patients had hypocholic stools and were evaluated rapidly to exclude biliary atresia, which was the final diagnosis in 74 (59%). Overall, 50 patients underwent genetic testing, 6 with single confirmatory gene testing, 38 through panels, and 6 with trio whole-exome sequencing because of complex phenotype. The genetic testing detection rate was 60%: the final diagnosis was Alagille syndrome in 11, progressive familial intrahepatic cholestasis type 2 in 6, alpha-1-antitrypsin deficiency in 3, and progressive familial intrahepatic cholestasis type 3 in 2; a further 7 genetic conditions were identified in 1 child each. Overall, only 18 of 125 (14%) remained with an indeterminate etiology. CONCLUSIONS: This protocol combining clinical and genetic assessment proved to be an effective diagnostic tool for neonatal/infantile cholestasis, identifying inherited disorders with a high detection rate. It also could allow a noninvasive diagnosis in children presenting with colored stools.