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The minichromosome maintenance (MCM) complex acts as a DNA helicase during DNA replication, and thereby regulates cell cycle progression and proliferation. In addition, MCM-complex components localize to centrosomes and play an independent role in ciliogenesis. Pathogenic variants in genes coding for MCM components and other DNA replication factors have been linked to growth and developmental disorders as Meier–Gorlin syndrome and Seckel syndrome. Trio exome/genome sequencing identified the same de novo MCM6 missense variant p.(Cys158Tyr) in two unrelated individuals that presented with overlapping phenotypes consisting of intra-uterine growth retardation, short stature, congenital microcephaly, endocrine features, developmental delay and urogenital anomalies. The identified variant affects a zinc binding cysteine in the MCM6 zinc finger signature. This domain, and specifically cysteine residues, are essential for MCM-complex dimerization and the induction of helicase activity, suggesting a deleterious effect of this variant on DNA replication. Fibroblasts derived from the two affected individuals showed defects both in ciliogenesis and cell proliferation. We additionally traced three unrelated individuals with de novo MCM6 variants in the oligonucleotide binding (OB)-fold domain, presenting with variable (neuro)developmental features including autism spectrum disorder, developmental delay, and epilepsy. Taken together, our findings implicate de novo MCM6 variants in neurodevelopmental disorders. The clinical features and functional defects related to the zinc binding residue resemble those observed in syndromes related to other MCM components and DNA replication factors, while de novo OB-fold domain missense variants may be associated with more variable neurodevelopmental phenotypes. These data encourage consideration of MCM6 variants in the diagnostic arsenal of NDD.

Cirrhosis is usually a late-onset and life-threatening disease characterized by fibrotic scarring and inflammation that disrupts liver architecture and function. While it is typically the result of alcoholism or hepatitis viral infection in adults, its etiology in infants is much less understood. In this study, we report 14 children from ten unrelated families presenting with a syndromic form of pediatric liver cirrhosis. By genome/exome sequencing, we found recessive variants in FOCAD segregating with the disease. Zebrafish lacking focad phenocopied the human disease, revealing a signature of altered messenger RNA (mRNA) degradation processes in the liver. Using patient’s primary cells and CRISPR-Cas9-mediated inactivation in human hepatic cell lines, we found that FOCAD deficiency compromises the SKI mRNA surveillance pathway by reducing the levels of the RNA helicase SKIC2 and its cofactor SKIC3. FOCAD knockout hepatocytes exhibited lowered albumin expression and signs of persistent injury accompanied by CCL2 overproduction. Our results reveal the importance of FOCAD in maintaining liver homeostasis and disclose a possible therapeutic intervention point via inhibition of the CCL2/CCR2 signaling axis.

Purpose Within this study, we aimed to discover novel gene–disease associations in patients with no genetic diagnosis after exome/genome sequencing (ES/GS). Methods We followed two approaches: (1) a patient-centered approach, which after routine diagnostic analysis systematically interrogates variants in genes not yet associated to human diseases; and (2) a gene variant centered approach. For the latter, we focused on de novo variants in patients that presented with neurodevelopmental delay (NDD) and/or intellectual disability (ID), which are the most common reasons for genetic testing referrals. Gene–disease association was assessed using our data repository that combines ES/GS data and Human Phenotype Ontology terms from over 33,000 patients. Results We propose six novel gene–disease associations based on 38 patients with variants in the BLOC1S1, IPO8, MMP15, PLK1, RAP1GDS1, and ZNF699 genes. Furthermore, our results support causality of 31 additional candidate genes that had little published evidence and no registered OMIM phenotype (56 patients). The phenotypes included syndromic/nonsyndromic NDD/ID, oral–facial–digital syndrome, cardiomyopathies, malformation syndrome, short stature, skeletal dysplasia, and ciliary dyskinesia. Conclusion Our results demonstrate the value of data repositories which combine clinical and genetic data for discovering and confirming gene–disease associations. Genetic laboratories should be encouraged to pursue such analyses for the benefit of undiagnosed patients and their families.

Biallelic changes in the ZNFX1 gene have been recently reported to cause severe familial immunodeficiency. Through a search of our bio/databank with information from genetic testing of >55 000 individuals, we identified nine additional patients from seven families with six novel homozygous ZNFX1 variants. Consistent with the previously described phenotype, our patients suffered from monocytosis, thrombocytopenia, hepatosplenomegaly, recurrent infections, and lymphadenopathy. The two most severely affected probands also had renal involvement and clinical presentations compatible with hemophagocytic lymphohistiocytosis. The disease was less lethal among our patients than previously reported. We identified two missense changes, two variants predicted to result in complete protein loss through nonsense-mediated decay, and two frameshift changes that likely introduce a truncation. Our findings (i) independently confirm the role of ZNFX1 in primary genetic immunodeficiency, (ii) expand the genetic and clinical spectrum of ZNFX1-related disease, and (iii) illustrate the utility of large, well-curated, and continually updated genotype-phenotype databases in resolving molecular diagnoses of patients with initially negative genetic testing findings.

Recently, ADAMTS19 was identified as a novel causative gene for autosomal recessive heart valve disease (HVD), affecting mainly the aortic and pulmonary valves. Exome sequencing and data repository (CentoMD) analyses were performed to identify patients with ADAMTS19 variants (two families). A third family was recognized based on cardiac phenotypic similarities and SNP array homozygosity. Three novel loss of function (LoF) variants were identified in six patients from three families. Clinically, all patients presented anomalies of the aortic/pulmonary valves, which included thickening of valve leaflets, stenosis and insufficiency. Three patients had (recurrent) subaortic membrane, suggesting that ADAMTS19 is the first gene identified related to discrete subaortic stenosis. One case presented a bi-commissural pulmonary valve. All patients displayed some degree of atrioventricular valve insufficiency. Other cardiac anomalies included atrial/ventricular septal defects, persistent ductus arteriosus, and mild dilated ascending aorta. Our findings confirm that biallelic LoF variants in ADAMTS19 are causative of a specific and recognizable cardiac phenotype. We recommend considering ADAMTS19 genetic testing in all patients with multiple semilunar valve abnormalities, particularly in the presence of subaortic membrane. ADAMTS19 screening in patients with semilunar valve abnormalities is needed to estimate the frequency of the HVD related phenotype, which might be not so rare.

Sphingomyelinases generate ceramide from sphingomyelin as a second messenger in intracellular signaling pathways involved in cell proliferation, differentiation, or apoptosis. Children from 12 unrelated families presented with microcephaly, simplified gyral pattern of the cortex, hypomyelination, cerebellar hypoplasia, congenital arthrogryposis, and early fetal/postnatal demise. Genomic analysis revealed bi-allelic loss-of-function variants in SMPD4, coding for the neutral sphingomyelinase-3 (nSMase-3/SMPD4). Overexpression of human Myc-tagged SMPD4 showed localization both to the outer nuclear envelope and the ER and additionally revealed interactions with several nuclear pore complex proteins by proteomics analysis. Fibroblasts from affected individuals showed ER cisternae abnormalities, suspected for increased autophagy, and were more susceptible to apoptosis under stress conditions, while treatment with siSMPD4 caused delayed cell cycle progression. Our data show that SMPD4 links homeostasis of membrane sphingolipids to cell fate by regulating the cross-talk between the ER and the outer nuclear envelope, while its loss reveals a pathogenic mechanism in microcephaly.

Human C2orf69 is an evolutionarily conserved gene whose function is unknown. Here, we report eight unrelated families from which 20 children presented with a fatal syndrome consisting of severe autoinflammation and progredient leukoencephalopathy with recurrent seizures; 12 of these subjects, whose DNA was available, segregated homozygous loss-of-function C2orf69 variants. C2ORF69 bears homology to esterase enzymes, and orthologs can be found in most eukaryotic genomes, including that of unicellular phytoplankton. We found that endogenous C2ORF69 (1) is loosely bound to mitochondria, (2) affects mitochondrial membrane potential and oxidative respiration in cultured neurons, and (3) controls the levels of the glycogen branching enzyme 1 (GBE1) consistent with a glycogen-storage-associated mitochondriopathy. We show that CRISPR-Cas9-mediated inactivation of zebrafish C2orf69 results in lethality by 8 months of age due to spontaneous epileptic seizures, which is preceded by persistent brain inflammation. Collectively, our results delineate an autoinflammatory Mendelian disorder of C2orf69 deficiency that disrupts the development/homeostasis of the immune and central nervous systems.