Histone modifications direct chromatin-templated events in the genome and regulate access to DNA sequence information. There are multiple types of modifications, and a common feature is their dynamic nature. An essential step for understanding their regulation, therefore, lies in characterizing the enzymes responsible for adding and removing histone modifications. Starting with a dosage-suppressor screen in Saccharomyces cerevisiae, we have discovered a functional interaction between the acetyltransferase Gcn5 and the protein phosphatase 2A (PP2A) complex, two factors that regulate post-translational modifications. We find that RTS1, one of two genes encoding PP2A regulatory subunits, is a robust and specific high-copy suppressor of temperature sensitivity of gcn5∆ and a subset of other gcn5∆ phenotypes. Conversely, loss of both PP2A(Rts1) and Gcn5 function in the SAGA and SLIK/SALSA complexes is lethal. RTS1 does not restore global transcriptional defects in gcn5∆; however, histone gene expression is restored, suggesting that the mechanism of RTS1 rescue includes restoration of specific cell cycle transcripts. Pointing to new mechanisms of acetylation-phosphorylation cross-talk, RTS1 high-copy rescue of gcn5∆ growth requires two residues of H2B that are phosphorylated in human cells. These data highlight the potential significance of dynamic phosphorylation and dephosphorylation of these deeply conserved histone residues for cell viability.

Congenital sodium diarrhea is a rare and life-threatening disorder characterized by a severe, secretory diarrhea containing high concentrations of sodium, leading to hyponatremia and metabolic acidosis. It may occur in isolation or in association with systemic features such as facial dysmorphism, choanal atresia, imperforate anus, and corneal erosions. Mutations in the serine protease inhibitor, Kunitz-Type 2 (SPINT2) gene have been associated with congenital sodium diarrhea and additional syndromic features. We present a child with congenital sodium diarrhea, cleft lip and palate, corneal erosions, optic nerve coloboma, and intermittent exotropia who was found to have biallelic mutations in SPINT2. One mutation, c.488A > G, predicting p.(Tyr163Cys), has been previously associated with a syndromic form of congenital sodium diarrhea. The other mutation, c.166_167dupTA, predicting p.(Asn57Thrfs*24) has not previously been reported and is likely a novel pathogenic variant for this disorder. We found only one other report of an optic nerve coloboma associated with SPINT2 mutations and this occurred in a patient with congenital tufting enteropathy. Our patient confirms an association of ocular coloboma with presumed loss of SPINT2 function.

Synaptic mitochondria are thought to be critical in supporting neuronal energy requirements at the synapse, and bioenergetic failure at the synapse may impair neural transmission and contribute to neurodegeneration. However, little is known about the energy requirements of synaptic vesicle release or whether these energy requirements go unmet in disease, primarily due to a lack of appropriate tools and sensitive assays. To determine the dependence of synaptic vesicle cycling on mitochondrially derived ATP levels, we developed two complementary assays sensitive to mitochondrially derived ATP in individual, living hippocampal boutons. The first is a functional assay for mitochondrially derived ATP that uses the extent of synaptic vesicle cycling as a surrogate for ATP level. The second uses ATP FRET sensors to directly measure ATP at the synapse. Using these assays, we show that endocytosis has high ATP requirements and that vesicle reacidification and exocytosis require comparatively little energy. We then show that to meet these energy needs, mitochondrially derived ATP is rapidly dispersed in axons, thereby maintaining near normal levels of ATP even in boutons lacking mitochondria. As a result, the capacity for synaptic vesicle cycling is similar in boutons without mitochondria as in those with mitochondria. Finally, we show that loss of a key respiratory subunit implicated in Leigh disease markedly decreases mitochondrially derived ATP levels in axons, thus inhibiting synaptic vesicle cycling. This proves that mitochondria-based energy failure can occur and be detected in individual neurons that have a genetic mitochondrial defect.

Cobalamin D deficiency (cblD) is one of the least common cobalamin metabolism disorders. It may result in isolated homocystinuria, isolated methylmalonic aciduria, or combined methylmalonic aciduria and homocystinuria (cblD-combined). Only seven cases of the combined cblD form have been reported to date. Due to the rarity of this disorder, the presentation and symptoms are not well described. We present an eighth case of the cblD-combined subtype, who had a positive newborn screen (NBS) on day of life 3. She was symptomatic and developed lethargy and poor oral intake at 8 days of life. She was treated with 10% dextrose, folinic acid, intramuscular hydroxocobalamin, and betaine. Despite the early initiation of treatment, she developed complications of the disease and was found to have abnormal brain imaging findings at 17 days of age and macular atrophy at 3 months of age and has global developmental delay. We provide detailed description of her presentation, her treatment, and her complications to aid in the understanding of this rare disorder, which is very similar to the more common cobalamin C disorder (cblC).

Synaptic mitochondria are thought to be critical in supporting neuronal energy requirements at the synapse, and bioenergetic failure at the synapse may impair neural transmission and contribute to neurodegeneration. However, little is known about the energy requirements of synaptic vesicle release or whether these energy requirements go unmet in disease, primarily due to a lack of appropriate tools and sensitive assays. To determine the dependence of synaptic vesicle cycling on mitochondrially derived ATP levels, we developed two complementary assays sensitive to mitochondrially derived ATP in individual, living hippocampal boutons. The first is a functional assay for mitochondrially derived ATP that uses the extent of synaptic vesicle cycling as a surrogate for ATP level. The second uses ATP FRET sensors to directly measure ATP at the synapse. Using these assays, we show that endocytosis has high ATP requirements and that vesicle reacidification and exocytosis require comparatively little energy. We then show that to meet these energy needs, mitochondrially derived ATP is rapidly dispersed in axons, thereby maintaining near normal levels of ATP even in boutons lacking mitochondria. As a result, the capacity for synaptic vesicle cycling is similar in boutons without mitochondria as in those with mitochondria. Finally, we show that loss of a key respiratory subunit implicated in Leigh disease markedly decreases mitochondrially derived ATP levels in axons, thus inhibiting synaptic vesicle cycling. This proves that mitochondria-based energy failure can occur and be detected in individual neurons that have a genetic mitochondrial defect.

Hunter syndrome, or mucopolysaccharidosis (MPS) II, is a rare lysosomal disorder characterized by progressive, multi-system disease. As most symptoms cannot be reversed once established, early detection and treatment prior to the onset of clinical symptoms are critical. However, it is difficult to identify affected individuals early in disease, and therefore the long-term outcomes of initiating treatment during this optimal time period are incompletely described. We report long-term clinical outcomes of treatment when initiated prior to obvious clinical signs by comparing the courses of two siblings with neuronopathic Hunter syndrome (c.1504 T > G[p.W502G]), one who was diagnosed due to clinical disease (Sibling-O, age 3.7 years) and the other who was diagnosed before disease was evident (Sibling-Y, age 12 months), due to his older sibling's findings. The brothers began enzyme replacement therapy within a month of diagnosis. Around the age of 5 years, Sibling-O had a cognitive measurement score in the impaired range of <55 (average range 85-115), whereas Sibling-Y at this age received a score of 91. Sibling-O has never achieved toilet training and needs direct assistance with toileting, dressing, and washing, while Sibling-Y is fully toilet-trained and requires less assistance with daily activities. Both siblings have demonstrated sensory-seeking behaviors, hyperactivity, impulsivity, and sleep difficulties; however, Sibling-O demonstrates physical behaviors that his brother does not, namely biting, pushing, and frequent elopement. Since the time of diagnosis, Sibling-O has had significant joint contractures and a steady deterioration in mobility leading to the need for an adaptive stroller at age 11, while Sibling-Y at age 10.5 could hike more than 6 miles without assistance. After nearly a decade of therapy, there were more severe and life-limiting disease manifestations for Sibling-O; data from caregiver interview indicated substantial differences in Quality of Life for the child and the family, dependent on timing of ERT. The findings from this sibling pair provide evidence of superior somatic and neurocognitive outcomes associated with presymptomatic treatment of Hunter syndrome, aligned with current considerations for newborn screening.

Disrupted energy metabolism drives cell dysfunction and disease, but approaches to increase or preserve ATP are lacking. To generate a comprehensive metabolic map of genes and pathways that regulate cellular ATP-the ATPome-we conducted a genome-wide CRISPR interference/activation screen integrated with an ATP biosensor. We show that ATP level is modulated by distinct mechanisms that promote energy production or inhibit consumption. In our system HK2 is the greatest ATP consumer, indicating energy failure may not be a general deficiency in producing ATP, but rather failure to recoup the ATP cost of glycolysis and diversion of glucose metabolites to the pentose phosphate pathway. We identify systems-level reciprocal inhibition between the HIF1 pathway and mitochondria; glycolysis-promoting enzymes inhibit respiration even when there is no glycolytic ATP production, and vice versa. Consequently, suppressing alternative metabolism modes paradoxically increases energy levels under substrate restriction. This work reveals mechanisms of metabolic control, and identifies therapeutic targets to correct energy failure.

Insufficient or dysregulated energy metabolism may underlie diverse inherited and degenerative diseases, cancer, and even aging itself. ATP is the central energy carrier in cells, but critical pathways for regulating ATP levels are not systematically understood. We combined a pooled clustered regularly interspaced short palindromic repeats interference (CRISPRi) library enriched for mitochondrial genes, a fluorescent biosensor, and fluorescence-activated cell sorting (FACS) in a high-throughput genetic screen to assay ATP concentrations in live human cells. We identified genes not known to be involved in energy metabolism. Most mitochondrial ribosomal proteins are essential in maintaining ATP levels under respiratory conditions, and impaired respiration predicts poor growth. We also identified genes for which coenzyme Q10 (CoQ10) supplementation rescued ATP deficits caused by knockdown. These included CoQ10 biosynthetic genes associated with human disease and a subset of genes not linked to CoQ10 biosynthesis, indicating that increasing CoQ10 can preserve ATP in specific genetic contexts. This screening paradigm reveals mechanisms of metabolic control and genetic defects responsive to energy-based therapies.

We describe an 11-year old boy with severe global developmental delays, failure to thrive and growth retardation, refractory seizures with recurrent status epilepticus, hypogammaglobulinemia, hypergonadotropic hypogonadism, and duodenal strictures. He had facial and skin findings compatible with trichothiodystrophy, including sparse and brittle hair, thin eyebrows, and dry skin. Exome sequencing showed a hemizygous, truncating variant in RNF113A, c.903_910delGCAGACCA, predicting p.(Gln302fs*12), that was inherited from his mother. Although his clinical features overlap closely with features described in the two previously reported male first cousins with RNF113A loss of function mutations, the duodenal strictures seen in this patient have not been reported. Interestingly, the patient's mother had short stature and 100% skewed X-inactivation as seen in other obligate female carriers. A second male with developmental delays, microcephaly, seizures, ambiguous genitalia, and facial anomalies that included sparse and brittle hair, thin eyebrows and dry skin was recently reported to have c.897_898delTG, predicting p.(Cys299*) in RNF113A and we provide additional clinical details for this patient. This report further supports deleterious variants in RNF113A as a cause of a novel trichothiodystrophy syndrome.

Background

Severe combined immunodeficiency (SCID) is characterized by arrested T-lymphocyte production and by B-lymphocyte dysfunction, which result in life-threatening infections. Early diagnosis of SCID through population-based screening of newborns can aid clinical management and help improve outcomes; it also permits the identification of previously unknown factors that are essential for lymphocyte development in humans.

Methods

SCID was detected in a newborn before the onset of infections by means of screening of T-cell-receptor excision circles, a biomarker for thymic output. On confirmation of the condition, the affected infant was treated with allogeneic hematopoietic stem-cell transplantation. Exome sequencing in the patient and parents was followed by functional analysis of a prioritized candidate gene with the use of human hematopoietic stem cells and zebrafish embryos.

Results

The infant had "leaky" SCID (i.e., a form of SCID in which a minimal degree of immune function is preserved), as well as craniofacial and dermal abnormalities and the absence of a corpus callosum; his immune deficit was fully corrected by hematopoietic stem-cell transplantation. Exome sequencing revealed a heterozygous de novo missense mutation, p.N441K, in BCL11B. The resulting BCL11B protein had dominant negative activity, which abrogated the ability of wild-type BCL11B to bind DNA, thereby arresting development of the T-cell lineage and disrupting hematopoietic stem-cell migration; this revealed a previously unknown function of BCL11B. The patient's abnormalities, when recapitulated in bcl11ba-deficient zebrafish, were reversed by ectopic expression of functionally intact human BCL11B but not mutant human BCL11B.

Conclusions

Newborn screening facilitated the identification and treatment of a previously unknown cause of human SCID. Coupling exome sequencing with an evaluation of candidate genes in human hematopoietic stem cells and in zebrafish revealed that a constitutional BCL11B mutation caused human multisystem anomalies with SCID and also revealed a prethymic role for BCL11B in hematopoietic progenitors. (Funded by the National Institutes of Health and others.).