De novo missense variants in exon 9 of SEPHS1 cause a neurodevelopmental condition with developmental delay, poor growth, hypotonia, and dysmorphic features.

Selenophosphate synthetase (SEPHS) plays an essential role in selenium metabolism. Two mammalian SEPHS paralogues, SEPHS1 and SEPHS2, share high sequence identity and structural homology with SEPHS. Here, we report nine individuals from eight families with developmental delay, growth and feeding problems, hypotonia, and dysmorphic features, all with heterozygous missense variants in SEPHS1. Eight of these individuals had a recurrent variant at amino acid position 371 of SEPHS1 (p.Arg371Trp, p.Arg371Gln, and p.Arg371Gly); seven of these variants were known to be de novo. Structural modeling and biochemical assays were used to understand the effect of these variants on SEPHS1 function. We found that a variant at residue Trp352 results in local structural changes of the C-terminal region of SEPHS1 that decrease the overall thermal stability of the enzyme. In contrast, variants of a solvent-exposed residue Arg371 do not impact enzyme stability and folding but could modulate direct protein-protein interactions of SEPSH1 with cellular factors in promoting cell proliferation and development. In neuronal SH-SY5Y cells, we assessed the impact of SEPHS1 variants on cell proliferation and ROS production and investigated the mRNA expression levels of genes encoding stress-related selenoproteins. Our findings provided evidence that the identified SEPHS1 variants enhance cell proliferation by modulating ROS homeostasis. Our study supports the hypothesis that SEPHS1 plays a critical role during human development and provides a basis for further investigation into the molecular mechanisms employed by SEPHS1. Furthermore, our data suggest that variants in SEPHS1 are associated with a neurodevelopmental disorder.

Expanding the genetic and phenotypic landscape of replication factor C complex-related disorders: RFC4 deficiency is linked to a multisystemic disorder.

The precise regulation of DNA replication is vital for cellular division and genomic integrity. Central to this process is the replication factor C (RFC) complex, encompassing five subunits, which loads proliferating cell nuclear antigen onto DNA to facilitate the recruitment of replication and repair proteins and enhance DNA polymerase processivity. While RFC1's role in cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is known, the contributions of RFC2-5 subunits on human Mendelian disorders is largely unexplored. Our research links bi-allelic variants in RFC4, encoding a core RFC complex subunit, to an undiagnosed disorder characterized by incoordination and muscle weakness, hearing impairment, and decreased body weight. We discovered across nine affected individuals rare, conserved, predicted pathogenic variants in RFC4, all likely to disrupt the C-terminal domain indispensable for RFC complex formation. Analysis of a previously determined cryo-EM structure of RFC bound to proliferating cell nuclear antigen suggested that the variants disrupt interactions within RFC4 and/or destabilize the RFC complex. Cellular studies using RFC4-deficient HeLa cells and primary fibroblasts demonstrated decreased RFC4 protein, compromised stability of the other RFC complex subunits, and perturbed RFC complex formation. Additionally, functional studies of the RFC4 variants affirmed diminished RFC complex formation, and cell cycle studies suggested perturbation of DNA replication and cell cycle progression. Our integrated approach of combining in silico, structural, cellular, and functional analyses establishes compelling evidence that bi-allelic loss-of-function RFC4 variants contribute to the pathogenesis of this multisystemic disorder. These insights broaden our understanding of the RFC complex and its role in human health and disease.

Targeted long-read sequencing identifies missing disease-causing variation.

Despite widespread clinical genetic testing, many individuals with suspected genetic conditions lack a precise diagnosis, limiting their opportunity to take advantage of state-of-the-art treatments. In some cases, testing reveals difficult-to-evaluate structural differences, candidate variants that do not fully explain the phenotype, single pathogenic variants in recessive disorders, or no variants in genes of interest. Thus, there is a need for better tools to identify a precise genetic diagnosis in individuals when conventional testing approaches have been exhausted. We performed targeted long-read sequencing (T-LRS) using adaptive sampling on the Oxford Nanopore platform on 40 individuals, 10 of whom lacked a complete molecular diagnosis. We computationally targeted up to 151 Mbp of sequence per individual and searched for pathogenic substitutions, structural variants, and methylation differences using a single data source. We detected all genomic aberrations-including single-nucleotide variants, copy number changes, repeat expansions, and methylation differences-identified by prior clinical testing. In 8/8 individuals with complex structural rearrangements, T-LRS enabled more precise resolution of the mutation, leading to changes in clinical management in one case. In ten individuals with suspected Mendelian conditions lacking a precise genetic diagnosis, T-LRS identified pathogenic or likely pathogenic variants in six and variants of uncertain significance in two others. T-LRS accurately identifies pathogenic structural variants, resolves complex rearrangements, and identifies Mendelian variants not detected by other technologies. T-LRS represents an efficient and cost-effective strategy to evaluate high-priority genes and regions or complex clinical testing results.

Mutations of the Transcriptional Corepressor ZMYM2 Cause Syndromic Urinary Tract Malformations.

Congenital anomalies of the kidney and urinary tract (CAKUT) constitute one of the most frequent birth defects and represent the most common cause of chronic kidney disease in the first three decades of life. Despite the discovery of dozens of monogenic causes of CAKUT, most pathogenic pathways remain elusive. We performed whole-exome sequencing (WES) in 551 individuals with CAKUT and identified a heterozygous de novo stop-gain variant in ZMYM2 in two different families with CAKUT. Through collaboration, we identified in total 14 different heterozygous loss-of-function mutations in ZMYM2 in 15 unrelated families. Most mutations occurred de novo, indicating possible interference with reproductive function. Human disease features are replicated in X. tropicalis larvae with morpholino knockdowns, in which expression of truncated ZMYM2 proteins, based on individual mutations, failed to rescue renal and craniofacial defects. Moreover, heterozygous Zmym2-deficient mice recapitulated features of CAKUT with high penetrance. The ZMYM2 protein is a component of a transcriptional corepressor complex recently linked to the silencing of developmentally regulated endogenous retrovirus elements. Using protein-protein interaction assays, we show that ZMYM2 interacts with additional epigenetic silencing complexes, as well as confirming that it binds to FOXP1, a transcription factor that has also been linked to CAKUT. In summary, our findings establish that loss-of-function mutations of ZMYM2, and potentially that of other proteins in its interactome, as causes of human CAKUT, offering new routes for studying the pathogenesis of the disorder.

The Impact of Rapid Exome Sequencing on Medical Management of Critically Ill Children.

OBJECTIVES: To evaluate the clinical usefulness of rapid exome sequencing (rES) in critically ill children with likely genetic disease using a standardized process at a single institution. To provide evidence that rES with should become standard of care for this patient population. STUDY DESIGN: We implemented a process to provide clinical-grade rES to eligible children at a single institution. Eligibility included (a) recommendation of rES by a consulting geneticist, (b) monogenic disorder suspected, (c) rapid diagnosis predicted to affect inpatient management, (d) pretest counseling provided by an appropriate provider, and (e) unanimous approval by a committee of 4 geneticists. Trio exome sequencing was sent to a reference laboratory that provided verbal report within 7-10 days. Clinical outcomes related to rES were prospectively collected. Input from geneticists, genetic counselors, pathologists, neonatologists, and critical care pediatricians was collected to identify changes in management related to rES. RESULTS: There were 54 patients who were eligible for rES over a 34-month study period. Of these patients, 46 underwent rES, 24 of whom (52%) had at least 1 change in management related to rES. In 20 patients (43%), a molecular diagnosis was achieved, demonstrating that nondiagnostic exomes could change medical management in some cases. Overall, 84% of patients were under 1 month old at rES request and the mean turnaround time was 9 days. CONCLUSIONS: rES testing has a significant impact on the management of critically ill children with suspected monogenic disease and should be considered standard of care for tertiary institutions who can provide coordinated genetics expertise.

SLC35A2-CDG: Functional characterization, expanded molecular, clinical, and biochemical phenotypes of 30 unreported Individuals.

Pathogenic de novo variants in the X-linked gene SLC35A2 encoding the major Golgi-localized UDP-galactose transporter required for proper protein and lipid glycosylation cause a rare type of congenital disorder of glycosylation known as SLC35A2-congenital disorders of glycosylation (CDG; formerly CDG-IIm). To date, 29 unique de novo variants from 32 unrelated individuals have been described in the literature. The majority of affected individuals are primarily characterized by varying degrees of neurological impairments with or without skeletal abnormalities. Surprisingly, most affected individuals do not show abnormalities in serum transferrin N-glycosylation, a common biomarker for most types of CDG. Here we present data characterizing 30 individuals and add 26 new variants, the single largest study involving SLC35A2-CDG. The great majority of these individuals had normal transferrin glycosylation. In addition, expanding the molecular and clinical spectrum of this rare disorder, we developed a robust and reliable biochemical assay to assess SLC35A2-dependent UDP-galactose transport activity in primary fibroblasts. Finally, we show that transport activity is directly correlated to the ratio of wild-type to mutant alleles in fibroblasts from affected individuals.

Identification of a deletion containing TBX4 in a neonate with acinar dysplasia by rapid exome sequencing.

We describe a neonate with severe respiratory failure due to acinar dysplasia found by rapid exome sequencing (rES), to have a deletion containing the TBX4 gene. rES can affect patient management in the intensive care unit and should be considered in concert with lung biopsy in neonates with undifferentiated respiratory failure.

Expanding phenotype with severe midline brain anomalies and missense variant supports a causal role for FOXA2 in 20p11.2 deletion syndrome.

Rare individuals with 20p11.2 proximal deletions have been previously reported, with a variable phenotype that includes heterotaxy, biliary atresia, midline brain defects associated with panhypopituitarism, intellectual disability, scoliosis, and seizures. Deletions have ranged in size from 277 kb to 11.96 Mb. We describe a newborn with a de novo 2.7 Mb deletion of 20p11.22p11.21 that partially overlaps previously reported deletions and encompasses FOXA2. Her clinical findings further expand the 20p11.2 deletion phenotype to include severe midline cranial and intracranial defects such as aqueductal stenosis with hydrocephalus, mesencephalosynapsis with diencephalic-mesencephalic junction dysplasia, and pyriform aperture stenosis. We also report one individual with a missense variant in FOXA2 who had abnormal glucose homeostasis, panhypopituitarism, and endodermal organ dysfunction. Together, these findings support the critical role of FOXA2 in panhypopituitarism and midline defects.

Modeling craniofacial and skeletal congenital birth defects to advance therapies.

Craniofacial development is an intricate process of patterning, morphogenesis, and growth that involves many tissues within the developing embryo. Genetic misregulation of these processes leads to craniofacial malformations, which comprise over one-third of all congenital birth defects. Significant advances have been made in the clinical management of craniofacial disorders, but currently very few treatments specifically target the underlying molecular causes. Here, we review recent studies in which modeling of craniofacial disorders in primary patient cells, patient-derived induced pluripotent stem cells (iPSCs), and mice have enhanced our understanding of the etiology and pathophysiology of these disorders while also advancing therapeutic avenues for their prevention.

The ATP-stimulated translocation promoter (ASTP) activity of glycerol kinase plays central role in adipogenesis.

Glycerol kinase (GK) is a multifunctional enzyme located at the interface of carbohydrate and fat metabolism. It contributes to both central carbon metabolism and adipogenesis; specifically, through its role as the ATP-stimulated translocation promoter (ASTP). GK overexpression leads to increased ASTP activity and increased fat storage in H4IIE cells. We performed metabolic flux analysis in human GK-overexpressing H4IIE cells and found that overexpressing cells had significantly altered fluxes through central carbon and lipid metabolism including increased flux through the pentose phosphate pathway and increased production of lipids. We also observed an equal contribution of glycerol to carbohydrate metabolism in all cell lines, suggesting that GK's alternate functions rather than its enzymatic function are important for these processes. To further elucidate the contributions of the enzymatic (phosphorylation) and alternative (ASTP) functions of GK in adipogenesis, we performed experiments on mammalian GK and E. coli GK. We determined that the ASTP function of GK (which is absent in E. coli GK) plays a greater role than the enzymatic activity in these processes. These studies further emphasize GK's diverse functionality and provides fundamental insights into the multiple protein functions of glycerol kinase.

Mandibulofacial Dysostosis with Microcephaly: Mutation and Database Update.

Mandibulofacial dysostosis with microcephaly (MFDM) is a multiple malformation syndrome comprising microcephaly, craniofacial anomalies, hearing loss, dysmorphic features, and, in some cases, esophageal atresia. Haploinsufficiency of a spliceosomal GTPase, U5-116 kDa/EFTUD2, is responsible. Here, we review the molecular basis of MFDM in the 69 individuals described to date, and report mutations in 38 new individuals, bringing the total number of reported individuals to 107 individuals from 94 kindreds. Pathogenic EFTUD2 variants comprise 76 distinct mutations and seven microdeletions. Among point mutations, missense substitutions are infrequent (14 out of 76; 18%) relative to stop-gain (29 out of 76; 38%), and splicing (33 out of 76; 43%) mutations. Where known, mutation origin was de novo in 48 out of 64 individuals (75%), dominantly inherited in 12 out of 64 (19%), and due to proven germline mosaicism in four out of 64 (6%). Highly penetrant clinical features include, microcephaly, first and second arch craniofacial malformations, and hearing loss; esophageal atresia is present in an estimated ∼27%. Microcephaly is virtually universal in childhood, with some adults exhibiting late "catch-up" growth and normocephaly at maturity. Occasionally reported anomalies, include vestibular and ossicular malformations, reduced mouth opening, atrophy of cerebral white matter, structural brain malformations, and epibulbar dermoid. All reported EFTUD2 mutations can be found in the EFTUD2 mutation database (http://databases.lovd.nl/shared/genes/EFTUD2).

Insulin sensitivity predictions in individuals with obesity and type II diabetes mellitus using mathematical model of the insulin signal transduction pathway.

Mathematical modeling approaches have been commonly used in complex signaling pathway studies such as the insulin signal transduction pathway. Our expanded mathematical model of the insulin signal transduction pathway was previously shown to effectively predict glucose clearance rates using mRNA levels of key components of the pathway in a mouse model. In this study, we re-optimized and applied our expanded model to study insulin sensitivity in other species and tissues (human skeletal muscle) with altered protein activities of insulin signal transduction pathway components. The model has now been optimized to predict the effect of short term exercise on insulin sensitivity for human test subjects with obesity or type II diabetes mellitus. A comparison between our extended model and the original model showed that our model better simulates the GLUT4 translocation events of the insulin signal transduction pathway and glucose uptake as a clinically relevant model output. Results from our extended model correlate with O'Gorman's published in-vivo results. This study demonstrates the ability to adapt this model to study insulin sensitivity to many biological systems (human skeletal muscle and mouse liver) with minimal changes in the model parameters.

Alcohol Intervention for Adolescents with Fetal Alcohol Spectrum Disorders: Project Step Up, a Treatment Development Study.

BACKGROUND: Project Step Up proposed to reduce alcohol consumption and alcohol-related negative outcomes in adolescents with fetal alcohol spectrum disorders (FASD). METHODS: The 54 participants (30 females, 24 males) were assigned to either Project Step Up Intervention (SUI) or Control conditions and were assessed prior to intervention, immediately following intervention, and at 3-month follow-up. Adolescents in the SUI condition participated in a 6-week, 60-minute group intervention that provided alcohol education and promoted adaptive responses to alcohol-related social pressures. Caregivers attended concurrent but separate sessions on the effects of prenatal alcohol exposure on the brain and how to handle parenting challenges associated with alcohol use in teens with FASD. RESULTS: Thirty-three percent (n = 18) of adolescents were classified as light/moderate drinkers, and 67% (n = 36) were abstinent/infrequent drinkers based on their lifetime drinking histories. Results revealed a significant decrease in self-reported alcohol risk and in alcohol-related negative behaviors (Cohen's d = 1.08 and 0.99) in light/moderate drinkers in the SUI compared to the Control group. These results were partially sustained at 3-month follow-up. Furthermore, adolescents in the abstinent/infrequent group exhibited no increase in alcohol-related outcomes suggesting that the group intervention used in this study was not iatrogenic. CONCLUSIONS: The success of this treatment development study provides preliminary support for effective treatment of adolescents with FASD to prevent or reduce alcohol use and its negative consequences in this high risk population.

Mathematical modeling of the insulin signal transduction pathway for prediction of insulin sensitivity from expression data.

Mathematical models of biological pathways facilitate a systems biology approach to medicine. However, these models need to be updated to reflect the latest available knowledge of the underlying pathways. We developed a mathematical model of the insulin signal transduction pathway by expanding the last major previously reported model and incorporating pathway components elucidated since the original model was reported. Furthermore, we show that inputting gene expression data of key components of the insulin signal transduction pathway leads to sensible predictions of glucose clearance rates in agreement with reported clinical measurements. In one set of simulations, our model predicted that glycerol kinase knockout mice have reduced GLUT4 translocation, and consequently, reduced glucose uptake. Additionally, a comparison of our extended model with the original model showed that the added pathway components improve simulations of glucose clearance rates. We anticipate this expanded model to be a useful tool for predicting insulin sensitivity in mammalian tissues with altered expression protein phosphorylation or mRNA levels of insulin signal transduction pathway components.

Duodenal atresia in 17q12 microdeletion including HNF1B: a new associated malformation in this syndrome.

Deletions of chromosome 17q12 [OMIM 614527] encompass a wide range of phenotypes, including renal cysts, diabetes mellitus, pancreatic structural abnormalities, genital tract anomalies, developmental delay, learning difficulties, and more recently, autism spectrum disorder and schizophrenia. To date, gastrointestinal malformations have not been fully characterized in this syndrome. In this case report, we describe a four-year-old girl with a 17q12 microdeletion who was born with duodenal atresia, bilateral renal cysts, left kidney dysplasia, a midline cystic structure at the conus medullaris, and dysmorphic features. Both the patient and her affected father were found to have a deletion of 17q12, which encompasses the HNF1B (hepatocyte nuclear factor beta). It is hypothesized that HNF1B may play a role in intestinal differentiation and development. Our clinical report further expands the pre-and post-natal presentation of this rare microdeletion syndrome.

Clinical exome sequencing for genetic identification of rare Mendelian disorders.

IMPORTANCE: Clinical exome sequencing (CES) is rapidly becoming a common molecular diagnostic test for individuals with rare genetic disorders. OBJECTIVE: To report on initial clinical indications for CES referrals and molecular diagnostic rates for different indications and for different test types. DESIGN, SETTING, AND PARTICIPANTS: Clinical exome sequencing was performed on 814 consecutive patients with undiagnosed, suspected genetic conditions at the University of California, Los Angeles, Clinical Genomics Center between January 2012 and August 2014. Clinical exome sequencing was conducted as trio-CES (both parents and their affected child sequenced simultaneously) to effectively detect de novo and compound heterozygous variants or as proband-CES (only the affected individual sequenced) when parental samples were not available. MAIN OUTCOMES AND MEASURES: Clinical indications for CES requests, molecular diagnostic rates of CES overall and for phenotypic subgroups, and differences in molecular diagnostic rates between trio-CES and proband-CES. RESULTS: Of the 814 cases, the overall molecular diagnosis rate was 26% (213 of 814; 95% CI, 23%-29%). The molecular diagnosis rate for trio-CES was 31% (127 of 410 cases; 95% CI, 27%-36%) and 22% (74 of 338 cases; 95% CI, 18%-27%) for proband-CES. In cases of developmental delay in children (<5 years, n = 138), the molecular diagnosis rate was 41% (45 of 109; 95% CI, 32%-51%) for trio-CES cases and 9% (2 of 23, 95% CI, 1%-28%) for proband-CES cases. The significantly higher diagnostic yield (P value = .002; odds ratio, 7.4 [95% CI, 1.6-33.1]) of trio-CES was due to the identification of de novo and compound heterozygous variants. CONCLUSIONS AND RELEVANCE: In this sample of patients with undiagnosed, suspected genetic conditions, trio-CES was associated with higher molecular diagnostic yield than proband-CES or traditional molecular diagnostic methods. Additional studies designed to validate these findings and to explore the effect of this approach on clinical and economic outcomes are warranted.

Dual diagnosis of UQCRFS1-related mitochondrial complex III deficiency and recessive GJA8-related cataracts.

Biallelic pathogenic variants in UQCRFS1 underlie a rare form of isolated mitochondrial complex III deficiency associated with lactic acidosis and a distinctive scalp alopecia previously described in two unrelated probands. Here, we describe a participant in the Undiagnosed Diseases Network (UDN) with a dual diagnosis of two autosomal recessive disorders revealed by genome sequencing: UQCRFS1-related mitochondrial complex III deficiency and GJA8-related cataracts. Both pathogenic variants have been reported before: UQCRFS1 (NM_006003.3:c.215-1 G>C, p.Val72_Thr81del10) in a case with mitochondrial complex III deficiency and GJA8 (NM 005267.5:c.736 G>T, p.Glu246*) as a somatic change in aged cornea leading to decreased junctional coupling. A multi-modal approach combining enzyme assays and cellular proteomics analysis provided clear evidence of complex III respiratory chain dysfunction and low abundance of the Rieske iron-sulfur protein, validating the pathogenic effect of the UQCRFS1 variant. This report extends the genotypic and phenotypic spectrum for these two rare disorders and highlights the utility of deep phenotyping and genomics data to achieve diagnosis and insights into rare disease.

Familial microdeletion of 17q24.3 upstream of SOX9 is associated with isolated Pierre Robin sequence due to position effect.

Pierre Robin sequence (PRS) is a malformation pattern characterized by the core triad of retrognathia, glossoptosis, and cleft palate that causes difficulty in glossopharyngeal-laryngeal-vagal functions. The etiology of PRS remains largely unknown; previous reports have suggested that it is caused by intrauterine constriction or external conditions such as oligohydramnios, breech position, or abnormal uterine anatomy. Genetic causes include occurrence as a manifestation of many single gene conditions and chromosomal rearrangements. Positional effect on some loci or genes, including SOX9 has also been posited as a cause. Here, we report on an 18-month-old girl born with isolated PRS. Clinical chromosome microarray analysis (CMA) revealed a maternally inherited ~623 kb microdeletion that is -725 kb upstream of 5' SOX9 at chromosome locus 17q24.3. Her mother had cleft palate. This region, although devoid of any genes, is known to have a position effect on SOX9 due to elimination of highly conserved non-coding cis-regulatory elements. This report supports the evidence that deregulation of an intact SOX9 coding region is a cause of or associated with isolated PRS, and provides further evidence that CMA in the clinical setting is a powerful tool in detecting microdeletions in gene "desert" regions that have pathogenic position effect on specific genes.

First report of a de novo 18q11.2 microdeletion including GATA6 associated with complex congenital heart disease and renal abnormalities.

Deletions of the long arm of chromosome 18 have been previously reported in many patients. Most cases involve the more distal regions of the long arm (18q21.1->qter). However, proximal interstitial deletions involving 18q11.2 are extremely rare. Here we report on a 14-month-old female with a 4.7 Mb (19,667,062-24,401,876 hg19) de novo interstitial deletion within chromosomal band 18q11.2, which includes GATA6 and 24 other RefSeq genes. The clinical features of our patient include complex congenital heart defects, a double outlet right ventricle, a subaortic ventricular septal defect, D-malposed great arteries, an atrial septal defect, a dysplastic aortic valve and patent ductus arteriosus. In addition, she had renal anomalies-a duplicated collecting system on the left and mild right hydronephrosis. These heart and renal defects are not reported in other patients with 18q proximal interstitial deletions. Heterozygous point mutations in GATA6, encoding for a zinc finger transcription factor, have been shown to cause congenital heart defects. Given the well-established biological role of GATA6 in cardiac development, a deletion of GATA6 is very likely responsible for our patient's complex congenital heart defects. This is the smallest and most proximal 18q11.2 deletion involving GATA6 that is associated with complex congenital heart disease and renal anomalies.