We report population findings from newborn screening for biotinidase deficiency in California, representing over 2,000,000 newborns. The incidence of profound deficiency was 1/73,629, higher than in other reported populations. Out of 28 patients with profound biotinidase deficiency, 19 were of Hispanic descent, suggesting an increased frequency among this group. Of the 28 patients, 23 underwent mutation analysis of the BTD gene, with one common mutation, 528G>T, found in 43.3% of Hispanic alleles tested.

Phosphomannomutase 2 deficiency (PMM2-CDG) is the most common congenital disorder of glycosylation (CDG). Hypoglycemia has been reported in various CDG including PMM2-CDG. The frequency and etiology of hypoglycemia in PMM2-CDG are not well studied.

Newborn screening is an incredibly useful tool for the early identification of many metabolic disorders, including fatty acid oxidation (FAO) disorders. In many cases, molecular tests are necessary to reach a final diagnosis, highlighting the need for a thorough evaluation of genes implicated in FAO disorders. Using the ClinGen (Clinical Genome Resource) clinical validity framework, thirty genes were analyzed for the strength of evidence supporting their association with FAO disorders. Evidence was gathered from the literature by biocurators and presented to disease experts for review in order to assign a clinical validity classification of Definitive, Strong, Moderate, Limited, Disputed, Refuted, or No Reported Evidence. Of the gene-disease relationships evaluated, 22/30 were classified as Definitive, three as Moderate, one as Limited, three as No Reported Evidence and one as Disputed. Gene-disease relationships with a Limited, Disputed, and No Reported Evidence were found on two, six, and up to four panels out of 30 FAO disorder-specific panels, respectively, in the National Institute of Health Genetic Testing Registry, while over 70% of the genes on panels are definitively associated with an FAO disorder. These results highlight the need to systematically assess the clinical relevance of genes implicated in fatty acid oxidation disorders in order to improve the interpretation of genetic testing results and diagnosis of patients with these disorders.

Mitochondrial DNA (mtDNA) depletion syndromes (MDSs) are a clinically and molecularly heterogeneous group of mitochondrial cytopathies characterized by severe mtDNA copy number reduction in affected tissues. Clinically, MDSs are mainly categorized as myopathic, encephalomyopathic, hepatocerebral, or multi-systemic forms. To date, the myopathic form of MDS is mainly caused by mutations in the TK2 gene, which encodes thymidine kinase 2, the first and rate limiting step enzyme in the phosphorylation of pyrimidine nucleosides. We analyzed 9 unrelated families with 11 affected subjects exhibiting the myopathic form of MDS, by sequencing the TK2 gene. Twelve mutations including 4 novel mutations were detected in 9 families. Skeletal muscle specimens were available from 7 out of 11 subjects. Respiratory chain enzymatic activities in skeletal muscle were measured in 6 subjects, and enzymatic activities were reduced in 3 subjects. Quantitative analysis of mtDNA content in skeletal muscle was performed in 5 subjects, and marked mtDNA content reduction was observed in each. In addition, we outline the molecular and clinical characteristics of this syndrome in a total of 52 patients including those previously reported, and a total of 36 TK2 mutations are summarized. Clinically, hypotonia and proximal muscle weakness are the major phenotypes present in all subjects. In summary, our study expands the molecular and clinical spectrum associated with TK2 deficiency.

The homeodomain zipper family (HD-ZIP) of transcription factors is present only in plants and plays important role in the regulation of plant-specific processes. The subfamily IV of HDZ transcription factors (HD-ZIP IV) has primarily been implicated in the regulation of epidermal structure development. Though this gene family is present in all lineages of land plants, members of this gene family have not been identified in banana, which is one of the major staple fruit crops. In the present work, we identified 21 HDZIV encoding genes in banana by the computational analysis of banana genome resource. Our analysis suggested that these genes putatively encode proteins having all the characteristic domains of HDZIV transcription factors. The phylogenetic analysis of the banana HDZIV family genes further confirmed that after separation from a common ancestor, the banana, and poales lineages might have followed distinct evolutionary paths. Further, we conclude that segmental duplication played a major role in the evolution of banana HDZIV encoding genes. All the identified banana HDZIV genes expresses in different banana tissue, however at varying levels. The transcript levels of some of the banana HDZIV genes were also detected in banana fruit pulp, suggesting their putative role in fruit attributes. A large number of genes of this family showed modulated expression under drought and salinity stress. Taken together, the present work lays a foundation for elucidation of functional aspects of the banana HDZIV encoding genes and for their possible use in the banana improvement programs.

Nuclear genes encode most mitochondrial proteins, and their mutations cause diverse and debilitating clinical disorders. To date, 1,200 of these mitochondrial genes have been recorded, while no standardized catalog exists of the associated clinical phenotypes. Such a catalog would be useful to develop methods to analyze human phenotypic data, to determine genotype-phenotype relations among many genes and diseases, and to support the clinical diagnosis of mitochondrial disorders. Here we establish a clinical phenotype catalog of 174 mitochondrial disease genes and study associations of diseases and genes. Phenotypic features such as clinical signs and symptoms were manually annotated from full-text medical articles and classified based on the hierarchical MeSH ontology. This classification of phenotypic features of each gene allowed for the comparison of diseases between different genes. In turn, we were then able to measure the phenotypic associations of disease genes for which we calculated a quantitative value that is based on their shared phenotypic features. The results showed that genes sharing more similar phenotypes have a stronger tendency for functional interactions, proving the usefulness of phenotype similarity values in disease gene network analysis. We then constructed a functional network of mitochondrial genes and discovered a higher connectivity for non-disease than for disease genes, and a tendency of disease genes to interact with each other. Utilizing these differences, we propose 168 candidate genes that resemble the characteristic interaction patterns of mitochondrial disease genes. Through their network associations, the candidates are further prioritized for the study of specific disorders such as optic neuropathies and Parkinson disease. Most mitochondrial disease phenotypes involve several clinical categories including neurologic, metabolic, and gastrointestinal disorders, which might indicate the effects of gene defects within the mitochondrial system. The accompanying knowledgebase (http://www.mitophenome.org/) supports the study of clinical diseases and associated genes.

Mitochondrial diseases are a clinically heterogenous group of disorders caused by respiratory chain dysfunction and associated with progressive, multi-systemic phenotype. There is no effective treatment or cure, and no FDA-approved drug for treating mitochondrial disease. To identify and characterize potential therapeutic compounds, we developed an in vitro screening assay and identified a group of direct AMP-activated protein kinase (AMPK) activators originally developed for the treatment of diabetes and metabolic syndrome. Unlike previously investigated AMPK agonists such as AICAR, these compounds allosterically activate AMPK in an AMP-independent manner, thereby increasing specificity and decreasing pleiotropic effects. The direct AMPK activator PT1 significantly improved mitochondrial function in assays of cellular respiration, energy status, and cellular redox. PT1 also protected against retinal degeneration in a mouse model of photoreceptor degeneration associated with mitochondrial dysfunction and oxidative stress, further supporting the therapeutic potential of AMP-independent AMPK agonists in the treatment of mitochondrial disease.

Stevia rebaudiana Bertoni is a commercially important zero calorie natural-sweetener herb which produce sweet compounds known as steviol glycosides. Rising demands of steviol glycosides by food and beverage industries has led to an increase in its cultivation in various countries. Unfortunately, stevia cultivation faces 2-25% yield penalty due to weeds which further adds to its cultivation cost. To resolve this major challenge, Agrobacterium-mediated genetic transformation of in vitro derived stevia-nodal explants using herbicide resistance gene (bar) has been optimized, for the production of stable transgenic stevia plants. Several parameters including explant type, pre-incubation duration, acetosyringone (As) concentration, Agrobacterium cell density, Agro-inoculation duration, co-cultivation duration, selection regime and plant growth regulators (PGRs) combination and concentration, have been successfully optimized. Among the two types of explants used, nodal explants showed a higher regeneration response of 82.85%, with an average of 25 shoots/explant. The best PGRs combination and concentration for shoot-induction, shoot-elongation and root-induction was found to be 6-benzyladenine (1.0 mg l-1) + naphthalene acetic acid (0.5 mg l-1), gibberellic acid (1.0 mg l-1), and half-strength MS medium, respectively. The two-step selection (phosphinothricin) regime resulted in an average transformation efficiency of 40.48% with nodal explants. Molecular characterization of putative transformants through PCR, RT-PCR, qRT-PCR and Southern-blot hybridization confirmed the presence, stability, expression as well as copy number of bar gene respectively. Compared to the non-transgenic plants, the T0 transgenic plants successfully tolerated 8 mg l-1 glufosinate ammonium sprays. Thus, the optimized protocol can be useful for the introduction of other genes (inter-kingdom transfer) into stevia genome.

Chromodomain helicase DNA-binding protein 4 (CHD4) is an ATP-dependent chromatin remodeler involved in epigenetic regulation of gene transcription, DNA repair, and cell cycle progression. Also known as Mi2β, CHD4 is an integral subunit of a well-characterized histone deacetylase complex. Here we report five individuals with de novo missense substitutions in CHD4 identified through whole-exome sequencing and web-based gene matching. These individuals have overlapping phenotypes including developmental delay, intellectual disability, hearing loss, macrocephaly, distinct facial dysmorphisms, palatal abnormalities, ventriculomegaly, and hypogonadism as well as additional findings such as bone fusions. The variants, c.3380G>A (p.Arg1127Gln), c.3443G>T (p.Trp1148Leu), c.3518G>T (p.Arg1173Leu), and c.3008G>A, (p.Gly1003Asp) (GenBank: NM_001273.3), affect evolutionarily highly conserved residues and are predicted to be deleterious. Previous studies in yeast showed the equivalent Arg1127 and Trp1148 residues to be crucial for SNF2 function. Furthermore, mutations in the same positions were reported in malignant tumors, and a de novo missense substitution in an equivalent arginine residue in the C-terminal helicase domain of SMARCA4 is associated with Coffin Siris syndrome. Cell-based studies of the p.Arg1127Gln and p.Arg1173Leu mutants demonstrate normal localization to the nucleus and HDAC1 interaction. Based on these findings, the mutations potentially alter the complex activity but not its formation. This report provides evidence for the role of CHD4 in human development and expands an increasingly recognized group of Mendelian disorders involving chromatin remodeling and modification.

In December 2014, a workshop entitled "Nutritional Interventions in Primary Mitochondrial Disorders: Developing an Evidence Base" was convened at the NIH with the goals of exploring the use of nutritional interventions in primary mitochondrial disorders (PMD) and identifying knowledge gaps regarding their safety and efficacy; identifying research opportunities; and forging collaborations among researchers, clinicians, patient advocacy groups, and federal partners. Sponsors included the NIH, the Wellcome Trust, and the United Mitochondrial Diseases Foundation. Dietary supplements have historically been used in the management of PMD due to their potential benefits and perceived low risk, even though little evidence exists regarding their effectiveness. PMD are rare and clinically, phenotypically, and genetically heterogeneous. Thus patient recruitment for randomized controlled trials (RCTs) has proven to be challenging. Only a few RCTs examining dietary supplements, singly or in combination with other vitamins and cofactors, are reported in the literature. Regulatory issues pertaining to the use of dietary supplements as treatment modalities further complicate the research and patient access landscape. As a preface to exploring a research agenda, the workshop included presentations and discussions on what PMD are; how nutritional interventions are used in PMD; challenges and barriers to their use; new technologies and approaches to diagnosis and treatment; research opportunities and resources; and perspectives from patient advocacy, industry, and professional organizations. Seven key areas were identified during the workshop. These areas were: 1) defining the disease, 2) clinical trial design, 3) biomarker selection, 4) mechanistic approaches, 5) challenges in using dietary supplements, 6) standards of clinical care, and 7) collaboration issues. Short- and long-term goals within each of these areas were identified. An example of an overarching goal is the enrollment of all individuals with PMD in a natural history study and a patient registry to enhance research capability. The workshop demonstrates an effective model for fostering and enhancing collaborations among NIH and basic research, clinical, patient, pharmaceutical industry, and regulatory stakeholders in the mitochondrial disease community to address research challenges on the use of dietary supplements in PMD.

Phytoene synthase (PSY) regulates the first committed step of the carotenoid biosynthetic pathway in plants. The present work reports identification and characterization of the three PSY genes (TaPSY1, TaPSY2 and TaPSY3) in wheat (Triticum aestivum L.). The TaPSY1, TaPSY2, and TaPSY3 genes consisted of three homoeologs on the long arm of group 7 chromosome (7L), short arm of group 5 chromosome (5S), and long arm of group 5 chromosome (5L), respectively in each subgenomes (A, B, and D) with a similarity range from 89% to 97%. The protein sequence analysis demonstrated that TaPSY1 and TaPSY3 retain most of conserved motifs for enzyme activity. Phylogenetic analysis of all TaPSY revealed an evolutionary relationship among PSY proteins of various monocot species. TaPSY derived from A and D subgenomes shared proximity to the PSY of Triticum urartu and Aegilops tauschii, respectively. The differential expression of TaPSY1, TaPSY2, and TaPSY3 in the various tissues, seed development stages, and stress treatments suggested their role in plant development, and stress condition. TaPSY3 showed higher expression in all tissues, followed by TaPSY1. The presence of multiple stress responsive cis-regulatory elements in promoter region of TaPSY3 correlated with the higher expression during drought and heat stresses has suggested their role in these conditions. The expression pattern of TaPSY3 was correlated with the accumulation of β-carotene in the seed developmental stages. Bacterial complementation assay has validated the functional activity of each TaPSY protein. Hence, TaPSY can be explored in developing genetically improved wheat crop.

DNA polymorphic markers and self-defined ethnicity groupings are used to group individuals with shared ancient geographic ancestry. Here we studied whether ancestral relationships between individuals could be identified from metabolic screening data reported by the California newborn screening (NBS) program. NBS data includes 41 blood metabolites measured by tandem mass spectrometry from singleton babies in 17 parent-reported ethnicity groupings. Ethnicity-associated differences identified for 71% of NBS metabolites (29 of 41, Cohen's d > 0.5) showed larger differences in blood levels of acylcarnitines than of amino acids (P < 1e-4). A metabolic distance measure, developed to compare ethnic groupings based on metabolic differences, showed low positive correlation with genetic and ancient geographic distances between the groups' ancestral world populations. Several outlier group pairs were identified with larger genetic and smaller metabolic distances (Black versus White) or with smaller genetic and larger metabolic distances (Chinese versus Japanese) indicating the influence of genetic and of environmental factors on metabolism. Using machine learning, comparison of metabolic profiles between all pairs of ethnic groupings distinguished individuals with larger genetic distance (Black versus Chinese, AUC = 0.96), while genetically more similar individuals could not be separated metabolically (Hispanic versus Native American, AUC = 0.51). Additionally, we identified metabolites informative for inferring metabolic ancestry in individuals from genetically similar populations, which included biomarkers for inborn metabolic disorders (C10:1, C12:1, C3, C5OH, Leucine-Isoleucine). This work sheds new light on metabolic differences in healthy newborns in diverse populations, which could have implications for improving genetic disease screening.

Improved second-tier tools are needed to reduce false-positive outcomes in newborn screening (NBS) for inborn metabolic disorders on the Recommended Universal Screening Panel (RUSP).

Low phytic acid is a trait desired in cereal crops and can be achieved by manipulating the genes involved either in its biosynthesis or its transport in the vacuoles. Previously, we have demonstrated that the wheat TaABCC13 protein is a functional transporter, primarily involved in heavy metal tolerance, and a probable candidate gene to achieve low phytate wheat. In the current study, RNA silencing was used to knockdown the expression of TaABCC13 in order to evaluate its functional importance in wheat. Transgenic plants with significantly reduced TaABCC13 transcripts in either seeds or roots were selected for further studies. Homozygous RNAi lines K1B4 and K4G7 exhibited 34-22% reduction of the phytic acid content in the mature grains (T4 seeds). These transgenic lines were defective for spike development, as characterized by reduced grain filling and numbers of spikelets. The seeds of transgenic wheat had delayed germination, but the viability of the seedlings was unaffected. Interestingly, early emergence of lateral roots was observed in TaABCC13-silenced lines as compared to non-transgenic lines. In addition, these lines also had defects in metal uptake and development of lateral roots in the presence of cadmium stress. Our results suggest roles of TaABCC13 in lateral root initiation and enhanced sensitivity towards heavy metals. Taken together, these data demonstrate that wheat ABCC13 is functionally important for grain development and plays an important role during detoxification of heavy metals.

Genitopatellar syndrome (GPS) is a skeletal dysplasia with cerebral and genital anomalies for which the molecular basis has not yet been determined. By exome sequencing, we found de novo heterozygous truncating mutations in KAT6B (lysine acetyltransferase 6B, formerly known as MYST4 and MORF) in three subjects; then by Sanger sequencing of KAT6B, we found similar mutations in three additional subjects. The mutant transcripts do not undergo nonsense-mediated decay in cells from subjects with GPS. In addition, human pathological analyses and mouse expression studies point to systemic roles of KAT6B in controlling organismal growth and development. Myst4 (the mouse orthologous gene) is expressed in mouse tissues corresponding to those affected by GPS. Phenotypic differences and similarities between GPS, the Say-Barber-Biesecker variant of Ohdo syndrome (caused by different mutations of KAT6B), and Rubinstein-Taybi syndrome (caused by mutations in other histone acetyltransferases) are discussed. Together, the data support an epigenetic dysregulation of the limb, brain, and genital developmental programs.

The purpose of this study was to enhance understanding of lysosomal acid lipase deficiency (LALD) in infancy.

PTEN-induced putative kinase 1 (PINK1) is a mitochondria-targeted kinase whose mutations are a cause of Parkinson's disease. We set out to better understand PINK1's effects on mitochondrial proteins in vivo. Using an unbiased phosphoproteomic screen in Drosophila, we found that PINK1 mediates the phosphorylation of MCAD, a mitochondrial matrix protein critical to fatty acid metabolism. By mimicking phosphorylation of this protein in a PINK1 null background, we restored PINK1 null's climbing, flight, thorax, and wing deficiencies. Owing to MCAD's role in fatty acid metabolism, we examined the metabolic profile of PINK1 null flies, where we uncovered significant disruptions in both acylcarnitines and amino acids. Some of these disruptions were rescued by phosphorylation of MCAD, consistent with MCAD's rescue of PINK1 null's organismal phenotypes. Our work validates and extends the current knowledge of PINK1, identifies a novel function of MCAD, and illuminates the need for and effectiveness of metabolic profiling in models of neurodegenerative disease.

Calcium/calmodulin-dependent protein kinase II (CAMK2) is one of the first proteins shown to be essential for normal learning and synaptic plasticity in mice, but its requirement for human brain development has not yet been established. Through a multi-center collaborative study based on a whole-exome sequencing approach, we identified 19 exceedingly rare de novo CAMK2A or CAMK2B variants in 24 unrelated individuals with intellectual disability. Variants were assessed for their effect on CAMK2 function and on neuronal migration. For both CAMK2A and CAMK2B, we identified mutations that decreased or increased CAMK2 auto-phosphorylation at Thr286/Thr287. We further found that all mutations affecting auto-phosphorylation also affected neuronal migration, highlighting the importance of tightly regulated CAMK2 auto-phosphorylation in neuronal function and neurodevelopment. Our data establish the importance of CAMK2A and CAMK2B and their auto-phosphorylation in human brain function and expand the phenotypic spectrum of the disorders caused by variants in key players of the glutamatergic signaling pathway.

Transcription factors BABY BOOM (BBM), WUSCHEL (WUS), BSD, LEAFY COTYLEDON (LEC), LEAFY COTYLEDON LIKE (LIL), VIVIPAROUS1 (VP1), CUP SHAPED COTYLEDONS (CUC), BOLITA (BOL), and AGAMOUS LIKE (AGL) play a crucial role in somatic embryogenesis. In this study, we identified eighteen genes of these nine transcription factors families from the banana genome database. All genes were analyzed for their structural features, subcellular, and chromosomal localization. Protein sequence analysis indicated the presence of characteristic conserved domains in these transcription factors. Phylogenetic analysis revealed close evolutionary relationship among most transcription factors of various monocots. The expression patterns of eighteen genes in embryogenic callus containing somatic embryos (precisely isolated by Laser Capture Microdissection), non-embryogenic callus, and cell suspension cultures of banana cultivar Grand Naine were analyzed. The application of 2, 4-dichlorophenoxyacetic acid (2, 4-D) in the callus induction medium enhanced the expression of MaBBM1, MaBBM2, MaWUS2, and MaVP1 in the embryogenic callus. It suggested 2, 4-D acts as an inducer for the expression of these genes. The higher expression of MaBBM2 and MaWUS2 in embryogenic cell suspension (ECS) as compared to non-embryogenic cells suspension (NECS), suggested that these genes may play a crucial role in banana somatic embryogenesis. MaVP1 showed higher expression in both ECS and NECS, whereas MaLEC2 expression was significantly higher in NECS. It suggests that MaLEC2 has a role in the development of non-embryogenic cells. We postulate that MaBBM2 and MaWUS2 can be served as promising molecular markers for the embryogencity in banana.

Intellectual disability (ID) and autism spectrum disorder (ASD) are genetically heterogeneous neurodevelopmental disorders. We sought to delineate the clinical, molecular, and neuroimaging spectrum of a novel neurodevelopmental disorder caused by variants in the zinc finger protein 292 gene (ZNF292).