Clinical and genetic delineation of autosomal recessive and dominant ACTL6B-related developmental brain disorders.

PURPOSE: This study aims to comprehensively delineate the phenotypic spectrum of ACTL6B-related disorders, previously associated with both autosomal recessive and autosomal dominant neurodevelopmental disorders. Molecularly, the role of the nucleolar protein ACTL6B in contributing to the disease has remained unclear. METHODS: We identified 105 affected individuals, including 39 previously reported cases, and systematically analysed detailed clinical and genetic data for all individuals. Additionally, we conducted knockdown experiments in neuronal cells to investigate the role of ACTL6B in ribosome biogenesis. RESULTS: Biallelic variants in ACTL6B are associated with severe-to-profound global developmental delay/intellectual disability (GDD/ID), infantile intractable seizures, absent speech, autistic features, dystonia, and increased lethality. De novo monoallelic variants result in moderate-to-severe GDD/ID, absent speech, and autistic features, while seizures and dystonia were less frequently observed. Dysmorphic facial features and brain abnormalities, including hypoplastic corpus callosum, parenchymal volume loss/atrophy, are common findings in both groups. We reveal that in the nucleolus, ACTL6B plays a crucial role in ribosome biogenesis, in particular in pre-rRNA processing. CONCLUSION: This study provides a comprehensive characterization of the clinical spectrum of both autosomal recessive and dominant forms of ACTL6B-associated disorders. It offers a comparative analysis of their respective phenotypes provides a plausible molecular explanation and suggests their inclusion within the expanding category of 'ribosomopathies'.

Variants in SCAF4 Cause a Neurodevelopmental Disorder and Are Associated with Impaired mRNA Processing.

RNA polymerase II interacts with various other complexes and factors to ensure correct initiation, elongation, and termination of mRNA transcription. One of these proteins is SR-related CTD-associated factor 4 (SCAF4), which is important for correct usage of polyA sites for mRNA termination. Using exome sequencing and international matchmaking, we identified nine likely pathogenic germline variants in SCAF4 including two splice-site and seven truncating variants, all residing in the N-terminal two thirds of the protein. Eight of these variants occurred de novo, and one was inherited. Affected individuals demonstrated a variable neurodevelopmental disorder characterized by mild intellectual disability, seizures, behavioral abnormalities, and various skeletal and structural anomalies. Paired-end RNA sequencing on blood lymphocytes of SCAF4-deficient individuals revealed a broad deregulation of more than 9,000 genes and significant differential splicing of more than 2,900 genes, indicating an important role of SCAF4 in mRNA processing. Knockdown of the SCAF4 ortholog CG4266 in the model organism Drosophila melanogaster resulted in impaired locomotor function, learning, and short-term memory. Furthermore, we observed an increased number of active zones in larval neuromuscular junctions, representing large glutamatergic synapses. These observations indicate a role of CG4266 in nervous system development and function and support the implication of SCAF4 in neurodevelopmental phenotypes. In summary, our data show that heterozygous, likely gene-disrupting variants in SCAF4 are causative for a variable neurodevelopmental disorder associated with impaired mRNA processing.

Heterozygous RNF13 Gain-of-Function Variants Are Associated with Congenital Microcephaly, Epileptic Encephalopathy, Blindness, and Failure to Thrive.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) initiates a stress response mechanism to clear out the unfolded proteins by either facilitating their re-folding or inducing their degradation. When this fails, an apoptotic cascade is initiated so that the affected cell is eliminated. IRE1α is a critical sensor of the unfolded-protein response, essential for initiating the apoptotic signaling. Here, we report an infantile neurodegenerative disorder associated with enhanced activation of IRE1α and increased apoptosis. Three unrelated affected individuals with congenital microcephaly, infantile epileptic encephalopathy, and profound developmental delay were found to carry heterozygous variants (c.932T>C [p.Leu311Ser] or c.935T>C [p.Leu312Pro]) in RNF13, which codes for an IRE1α-interacting protein. Structural modeling predicted that the variants, located on the surface of the protein, would not alter overall protein folding. Accordingly, the abundance of RNF13 and IRE1α was not altered in affected individuals' cells. However, both IRE1α-mediated stress signaling and stress-induced apoptosis were increased in affected individuals' cells. These results indicate that the RNF13 variants confer gain of function to the encoded protein and thereby lead to altered signaling of the ER stress response associated with severe neurodegeneration in infancy.

Variants in GNAI1 cause a syndrome associated with variable features including developmental delay, seizures, and hypotonia.

PURPOSE: Neurodevelopmental disorders (NDDs) encompass a spectrum of genetically heterogeneous disorders with features that commonly include developmental delay, intellectual disability, and autism spectrum disorders. We sought to delineate the molecular and phenotypic spectrum of a novel neurodevelopmental disorder caused by variants in the GNAI1 gene. METHODS: Through large cohort trio-based exome sequencing and international data-sharing, we identified 24 unrelated individuals with NDD phenotypes and a variant in GNAI1, which encodes the inhibitory Gαi1 subunit of heterotrimeric G-proteins. We collected detailed genotype and phenotype information for each affected individual. RESULTS: We identified 16 unique variants in GNAI1 in 24 affected individuals; 23 occurred de novo and 1 was inherited from a mosaic parent. Most affected individuals have a severe neurodevelopmental disorder. Core features include global developmental delay, intellectual disability, hypotonia, and epilepsy. CONCLUSION: This collaboration establishes GNAI1 variants as a cause of NDDs. GNAI1-related NDD is most often characterized by severe to profound delays, hypotonia, epilepsy that ranges from self-limiting to intractable, behavior problems, and variable mild dysmorphic features.

Medical Decision Support to Reduce Unwarranted Methylene Tetrahydrofolate Reductase (MTHFR) Genetic Testing.

Most major national medical associations have advised against routine MTHFR testing since at least 2013. However, many providers continue to order this unwarranted genetic test. This study assessed the efficacy of an electronic best practice alert to aid ordering providers. We tracked the rate of MTHFR tests ordered per 1 million patients in the twelve months before and after the implementation of an alert that suggested an alternative test. Associated factors including the ordering department, diagnosis, patient sex, and patient age were also analyzed. Chi square analysis was used to compare the difference between pre- and post-alert test ordering rates. A total of 997 MTHFR analysis were ordered in Southern California Kaiser Permanente from January 2017 through December 2018. Overall, the average MTHFR monthly test ordering rates dropped significantly from 12.93 per million patients in 2017 to 7.08 per million patients in 2018 (p = 0.0056). However, testing rates in children were unchanged and, in some associated diagnoses, such as psychiatric illnesses and neurodevelopmental conditions, the testing rates increased. Recommending an alternate test in lieu of the unwarranted one significantly reduced the overall rate of MTHFR testing. The alert was most effective for specialties and diagnoses where MTHFR was historically medically indicated. This suggests such alerts are an effective intervention that health care systems can implement to serve as an educational update and to reduce unwarranted genetic testing.

Clinical and radiographic delineation of Bent Bone Dysplasia-FGFR2 type or Bent Bone Dysplasia with Distinctive Clavicles and Angel-shaped Phalanges.

Bent Bone Dysplasia-FGFR2 type is a relatively recently described bent bone phenotype with diagnostic clinical, radiographic, and molecular characteristics. Here we report on 11 individuals, including the original four patients plus seven new individuals with three longer-term survivors. The prenatal phenotype included stillbirth, bending of the femora, and a high incidence of polyhydramnios, prematurity, and perinatal death in three of 11 patients in the series. The survivors presented with characteristic radiographic findings that were observed among those with lethality, including bent bones, distinctive (moustache-shaped) small clavicles, angel-shaped metacarpals and phalanges, poor mineralization of the calvarium, and craniosynostosis. Craniofacial abnormalities, hirsutism, hepatic abnormalities, and genitourinary abnormalities were noted as well. Longer-term survivors all needed ventilator support. Heterozygosity for mutations in the gene that encodes Fibroblast Growth Factor Receptor 2 (FGFR2) was identified in the nine individuals with available DNA. Description of these patients expands the prenatal and postnatal findings of Bent Bone Dysplasia-FGFR2 type and adds to the phenotypic spectrum among all FGFR2 disorders. © 2016 Wiley Periodicals, Inc.

Maximal dietary responsiveness after tetrahydrobiopterin (BH4) in 19 phenylalanine hydroxylase deficiency patients: What super-responders can expect.

Background: Inherited phenylalanine hydroxylase deficiency, also known as phenylketonuria (PKU), causes poor growth and neurologic deficits in the untreated state. After ascertainment through newborn screen and dietary phenylalanine (Phe) restriction to achieve plasma Phe in the range of 120-360 µmol/L, these disease manifestations can be prevented. Poor compliance with protein restricted diets supported by medical food is typical in later years, beginning in the late toddler and teenage years. Pharmacologic doses of oral tetrahydrobiopterin (BH4; sapropterin dihydrochloride) is effective in reducing plasma Phe in about 40-50% of PKU patients but effectiveness is highly variable. Objective: To assess the maximal responsiveness to 20 mg/kg/day oral BH4 as it affects plasma Phe and dietary Phe allowance in PKU patients. Materials and methods: This was a single-center, retrospective observational study, combining case reports of individual patients. We reported an outcome of 85 patients with PKU who were trialed on BH4. Phe levels and dietary records of 19 BH4 "super-responders" were analyzed. Results: Overall, 63.5% of the patients (54/85) were considered BH4 responders. However, we quantitated the dietary liberalization of 19 of our responsive patients (35%), those with at least a 2-fold increase in dietary Phe and maintenance of plasma Phe in treatment range. In these "super-responders", the mean plasma Phe at baseline was 371 ± 237 µmol/L and decreased to 284 ± 273 µmol/L after 1 year on BH4. Mean dietary Phe tolerance increased significantly from 595 ± 256 to 2260 ± 1414 mg/day (p ≤0.0001), while maintaining mean plasma Phe levels within treatment range. Four patients no longer required dietary Phe restriction and could discontinue medical food. The majority of patients had at least one BH4-responsive genotype. Conclusion: This cohort demonstrates the maximally achievable dietary liberalization which some PKU patients may expect with BH4 therapy. Health benefits are considered to accrue in patients with increased intact protein.

Biochemical and molecular characteristics among infants with abnormal newborn screen for very-long-chain acyl-CoA dehydrogenase deficiency: A single center experience.

Objective: To define the biochemical and molecular characteristics and diagnostic outcomes of a large US cohort of VLCAD deficiency positive cases as detected by newborn screening (NBS) with MS:MS. This relatively common disorder of fatty acid oxidation is screened for in every state in America and often results in extensive testing of multiple samples to arrive at a diagnostic conclusion. Materials and methods: We compared NBS dried blood spot (DBS) acylcarnitine profile (ACP) C14, C14:1, C14:2, C14:1/C12:1 ratio and plasma C14, C14:1, C14:2, C14:1/C12:1, C14:1/C16 and C14:1/C2 ratios among true positive and false positive cases. Results of VLCAD enzyme analysis, molecular testing and fibroblast fatty acid oxidation probe assay were analyzed. Results: The presence of compound heterozygous or homozygous pathogenic variants, along with elevations of C14, C14:1 and C14:1/C12:1 ratio, identified 19 VLCAD deficiency cases. All were asymptomatic at most recent follow-up visits. The C14:1/C12:1 ratio in NBS-DBS ACP and plasma acylcarnitine profiles at follow-up (follow-up plasma ACP), is the most useful marker to differentiate between true and false positive cases. Among all cases with molecular analysis data available, approximately 56.7% had a single pathogenic mutation. Lymphocyte enzyme analysis (n = 61) was uninformative in 23% of cases studied. Conclusion: VLCAD deficiency NBS by MS:MS is highly effective at identifying asymptomatic affected infants. Our cohort showed that elevation of C14:1/C12:1, in both NBS DBS and plasma ACP, was informative in discriminating affected from unaffected individuals and contributes to improve the accuracy of confirmatory testing of infants with presumptive positive for VLCAD deficiency.

HSD10 disease in a female: A case report and review of literature.

HSD10 disease is a rare X-linked mitochondrial disorder caused by pathogenic variants in the HSD17B10 gene. The phenotype results from impaired 17ß-hydroxysteroid dehydrogenase 10 (17ß-HSD10) protein structure and function. HSD10 is a multifunctional protein involved in enzymatic degradation of isoleucine and branched-chain fatty acids, the metabolism of sex hormones and neurosteroids, as well as in regulating mitochondrial RNA maturation. HSD10 disease is characterised by progressive neurologic impairment. Disease onset is varied and includes neonatal-onset, infantile-onset and late-onset in males. Females can also be affected. Our index case is a 45-month-old female, who initially presented at 11 months of age with global developmental delay. She subsequently began to lose previously acquired cognitive and motor skills starting around 29 months of age. Brain MRI showed abnormalities in the basal ganglia indicative of possible mitochondrial disease. Urine organic acid analysis revealed elevations of 2-methyl-3-hydroxybutyric acid and tiglyglycine. HSD17B10 gene sequencing revealed a likely pathogenic variant, NM_001037811.2:c.439C>T (p.Arg147Cys) inherited from her mother, expected to be causative of HSD10 disease. Her X-chromosome inactivation study is consistent with a skewed X-inactivation pattern. We report a female patient with HSD10 disease caused by a missense pathogenic variant, Arg147Cys in the HSD17B10 gene. The patient is the fifth severely affected female with this disease. This case adds to the small number of known affected families with this highly variable disease in the literature. These findings support the possibility of X-inactivation patterns influencing the penetrance of HSD10 disease in females.

Angiotensin II-accelerated atherosclerosis and aneurysm formation is attenuated in osteopontin-deficient mice.

Osteopontin (OPN) is expressed in atherosclerotic lesions, particularly in diabetic patients. To determine the role of OPN in atherogenesis, ApoE-/-OPN+/+, ApoE-/-OPN+/-, and ApoE-/-OPN-/- mice were infused with Ang II, inducing vascular OPN expression and accelerating atherosclerosis. Compared with ApoE-/-OPN+/+ mice, ApoE-/-OPN+/- and ApoE-/-OPN-/- mice developed less Ang II-accelerated atherosclerosis. ApoE-/- mice transplanted with bone marrow derived from ApoE-/-OPN-/- mice had less Ang II-induced atherosclerosis compared with animals receiving ApoE-/-OPN+/+ cells. Aortae from Ang II-infused ApoE-/-OPN-/- mice expressed less CD68, C-C-chemokine receptor 2, and VCAM-1. In response to intraperitoneal thioglycollate, recruitment of leukocytes in OPN-/- mice was impaired, and OPN-/- leukocytes exhibited decreased basal and MCP-1-directed migration. Furthermore, macrophage viability in atherosclerotic lesions from Ang II-infused ApoE-/-OPN-/- mice was decreased. Finally, Ang II-induced abdominal aortic aneurysm formation in ApoE-/-OPN-/- mice was reduced and associated with decreased MMP-2 and MMP-9 activity. These data suggest an important role for leukocyte-derived OPN in mediating Ang II-accelerated atherosclerosis and aneurysm formation.

2q23.1 microdeletion of the MBD5 gene in a female with seizures, developmental delay and distinct dysmorphic features.

We report a 2-year-old female who initially presented with seizures, developmental delay and dysmorphic features and was found to have a 0.3 Mb deletion at chromosome 2q23.1 encompassing the critical seizure gene, MBD5. Her distinct physical features include bifrontal narrowing with brachycephaly, low anterior hairline, hypotonic facial features with short upturned nose, flat nasal bridge, hypertelorism, tented upper lip with everted lower lip, downturned corners of her mouth, and relatively coarse facial features including thickened tongue. She also had a short neck, brachytelephalangy, clinodactyly, and hypertrichosis. At 3½ years she developed progressive ataxia and lost vocabulary at the age of 4. Regression has been reported in one other case of MBD5 deletion. MBD5 is a member of the methyl binding gene family and appears to be responsible for regulating DNA methylation in the central nervous system. Our patient was entirely deleted for the MBD5 gene with partial loss of the EPC2 gene, which suggests that haploinsufficiency of MBD5 is responsible for the distinct phenotype observed. This supports the hypothesis that MBD5 is indeed the critical gene implicated for the findings seen in patients with deletions of chromosome 2q23.1. Further studies are necessary to delineate the role that the MBD5 gene plays in the development of the brain and these specific physical characteristics.

2q23.1 microdeletion of the MBD5 gene in a female with seizures, developmental delay and distinct dysmorphic features.

We report a 2-year-old female who initially presented with seizures, developmental delay and dysmorphic features and was found to have a 0.3 Mb deletion at chromosome 2q23.1 encompassing the critical seizure gene, MBD5. Her distinct physical features include bifrontal narrowing with brachycephaly, low anterior hairline, hypotonic facial features with short upturned nose, flat nasal bridge, hypertelorism, tented upper lip with everted lower lip, downturned corners of her mouth, and relatively coarse facial features including thickened tongue. She also had a short neck, brachytelephalangy, clinodactyly, and hypertrichosis. At 3½ years she developed progressive ataxia and lost vocabulary at the age of 4. Regression has been reported in one other case of MBD5 deletion. MBD5 is a member of the methyl binding gene family and appears to be responsible for regulating DNA methylation in the central nervous system. Our patient was entirely deleted for the MBD5 gene with partial loss of the EPC2 gene, which suggests that haploinsufficiency of MBD5 is responsible for the distinct phenotype observed. This supports the hypothesis that MBD5 is indeed the critical gene implicated for the findings seen in patients with deletions of chromosome 2q23.1. Further studies are necessary to delineate the role that the MBD5 gene plays in the development of the brain and these specific physical characteristics.

Clinical review of genetic epileptic encephalopathies.

Seizures are a frequently encountered finding in patients seen for clinical genetics evaluations. The differential diagnosis for the cause of seizures is quite diverse and complex, and more than half of all epilepsies have been attributed to a genetic cause. Given the complexity of such evaluations, we highlight the more common causes of genetic epileptic encephalopathies and emphasize the usefulness of recent technological advances. The purpose of this review is to serve as a practical guide for clinical geneticists in the evaluation and counseling of patients with genetic epileptic encephalopathies. Common syndromes will be discussed, in addition to specific seizure phenotypes, many of which are refractory to anti-epileptic agents. Divided by etiology, we overview the more common causes of infantile epileptic encephalopathies, channelopathies, syndromic, metabolic, and chromosomal entities. For each condition, we will outline the diagnostic evaluation and discuss effective treatment strategies that should be considered.

Synthetic LXR ligand inhibits the development of atherosclerosis in mice.

The nuclear receptors LXRalpha and LXRbeta have been implicated in the control of cholesterol and fatty acid metabolism in multiple cell types. Activation of these receptors stimulates cholesterol efflux in macrophages, promotes bile acid synthesis in liver, and inhibits intestinal cholesterol absorption, actions that would collectively be expected to reduce atherosclerotic risk. However, synthetic LXR ligands have also been shown to induce lipogenesis and hypertriglyceridemia in mice, raising questions as to the net effects of these compounds on the development of cardiovascular disease. We demonstrate here that the nonsteroidal LXR agonist GW3965 has potent antiatherogenic activity in two different murine models. In LDLR(-/-) mice, GW3965 reduced lesion area by 53% in males and 34% in females. A similar reduction of 47% was observed in male apoE(-/-) mice. Long-term (12-week) treatment with LXR agonist had differential effects on plasma lipid profiles in LDLR(-/-) and apoE(-/-) mice. GW3965 induced expression of ATP-binding cassettes A1 and G1 in modified low-density lipoprotein-loaded macrophages in vitro as well as in the aortas of hyperlipidemic mice, suggesting that direct actions of LXR ligands on vascular gene expression are likely to contribute to their antiatherogenic effects. These observations provide direct evidence for an atheroprotective effect of LXR agonists and support their further evaluation as potential modulators of human cardiovascular disease.