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1.  Reduced Mitochondrial DNA Content and Heterozygous Nuclear Gene Mutations in Patients with Acute Liver Failure 
Historically, mitochondrial disorders have been associated with predominantly multisystem or neurological symptoms. If present, hepatic complications were thought to be a late feature. Recently, mutations in at least 4 nuclear genes have been identified in infants presenting with rapidly progressive hepatic failure which may be precipitated by infection or drugs. We aimed to determine if hepatic mitochondrial DNA (mtDNA) depletion is associated with apparently isolated hepatic failure in individuals with acute liver failure (ALF) of known or unknown etiologies undergoing liver transplant (LT) In addition we wished to establish if there was an excess of mutations in gene known to cause hepatic mtDNA depletion.
Using previously established methods, we demonstrated that end stage liver disease from known causes did not lead to hepatic mtDNA depletion. Using thresholds derived from ROC analysis, 66% of cases with ALF had probable or definite mtDNA depletion, including 34% with definite mtDNA depletion.
There was a small, but significant increase in the proportion of patients undergoing LT for ALF with heterozygous mutations known to lead to mtDNA depletion and hepatic failure compared with controls (P = 0.001).
Liver disease severe enough to require LT does not cause secondary mtDNA depletion. However, the majority of patients undergoing LT for ALF had reduced mtDNA content, which fell within the range seen in patients with classic mtDNA depletion. A subset of ALF patients have mutations in genes known to lead to mtDNA depletion and hepatic failure. Together, these results suggest defective mtDNA maintenance is associated with ALF.
PMCID: PMC4966813  PMID: 23783014
DGUOK, mitochondrial DNA depletion; next generation sequencing; POLG; Valproate
2.  SIGIRR Genetic Variants in Premature Infants With Necrotizing Enterocolitis 
Pediatrics  2015;135(6):e1530-e1534.
Necrotizing enterocolitis (NEC) is a severe form of bowel disease that develops in premature infants. Although animal data and human studies suggest that aberrant activation of the intestinal immune system contributes to NEC, the pathogenesis remains unclear. We hypothesized that inherited defects in the regulation of Toll-like receptor signaling can contribute to NEC susceptibility in premature infants. A forward genetic screen done in an infant with lethal NEC using exome sequencing identified a novel stop mutation (p.Y168X) and a rare missense variant (p.S80Y) in SIGIRR, a gene that inhibits intestinal Toll-like receptor signaling. Functional studies carried out in human embryonic kidney cells and intestinal epithelial cells demonstrated that SIGIRR inhibited inflammation induced by lipopolysaccharide, a cell wall component of Gram-negative bacteria implicated in NEC. The genetic variants identified in the infant with NEC resulted in loss of SIGIRR function and exaggerated inflammation in response to lipopolysaccharide. Additionally, Sanger sequencing identified missense, stop, or splice region SIGIRR variants in 10 of 17 premature infants with stage II+ NEC. To the best of our knowledge, this is one of the first reports of a phenotype associated with SIGIRR in humans. Our data provide novel mechanistic insight into the probable causation of NEC and support additional investigation of the hypothesis that inherited defects in the regulation of innate immune signaling can contribute to NEC susceptibility in premature infants.
PMCID: PMC4444800  PMID: 25963006
3.  Diversion of aspartate in ASS1-deficient tumors fosters de novo pyrimidine synthesis 
Nature  2015;527(7578):379-383.
PMCID: PMC4655447  PMID: 26560030
Argininosuccinate Synthase; Citrin; Warburg effect; pyrimidine synthesis; mTOR
4.  Protocol for the “Implementation, adoption, and utility of family history in diverse care settings” study 
Risk assessment with a thorough family health history is recommended by numerous organizations and is now a required component of the annual physical for Medicare beneficiaries under the Affordable Care Act. However, there are several barriers to incorporating robust risk assessments into routine care. MeTree, a web-based patient-facing health risk assessment tool, was developed with the aim of overcoming these barriers. In order to better understand what factors will be instrumental for broader adoption of risk assessment programs like MeTree in clinical settings, we obtained funding to perform a type III hybrid implementation-effectiveness study in primary care clinics at five diverse healthcare systems. Here, we describe the study’s protocol.
MeTree collects personal medical information and a three-generation family health history from patients on 98 conditions. Using algorithms built entirely from current clinical guidelines, it provides clinical decision support to providers and patients on 30 conditions. All adult patients with an upcoming well-visit appointment at one of the 20 intervention clinics are eligible to participate. Patient-oriented risk reports are provided in real time. Provider-oriented risk reports are uploaded to the electronic medical record for review at the time of the appointment. Implementation outcomes are enrollment rate of clinics, providers, and patients (enrolled vs approached) and their representativeness compared to the underlying population. Primary effectiveness outcomes are the percent of participants newly identified as being at increased risk for one of the clinical decision support conditions and the percent with appropriate risk-based screening. Secondary outcomes include percent change in those meeting goals for a healthy lifestyle (diet, exercise, and smoking). Outcomes are measured through electronic medical record data abstraction, patient surveys, and surveys/qualitative interviews of clinical staff.
This study evaluates factors that are critical to successful implementation of a web-based risk assessment tool into routine clinical care in a variety of healthcare settings. The result will identify resource needs and potential barriers and solutions to implementation in each setting as well as an understanding potential effectiveness.
Trial registration
PMCID: PMC4657284  PMID: 26597091
Risk stratification; Prevention; Primary care; Family health history
5.  Anti-Oxidant Response Genes sequence variants and BPD susceptibility in VLBW infants 
Pediatric research  2014;77(3):477-483.
Lung injury resulting from oxidative stress contributes to bronchopulmonary dysplasia (BPD) pathogenesis. Nuclear factor erythroid-2 related factor-2 (NFE2L2) regulates cytoprotective responses to oxidative stress by inducing enzymes containing anti-oxidant response elements (ARE). We hypothesized that ARE genetic variants will modulate susceptibility or severity of BPD in very low birth weight (VLBW) infants.
Blood samples obtained from VLBW infants were used for genotyping variants in the SOD2, NFE2L2, GCLC, GSTP1, HMOX1 and NQO1 genes. SNPs were genotyped utilizing TaqMan probes (Applied Biosystems (ABI), Grand Island, NY), and data was analyzed using the ABI HT7900. Genetic dominance and recessive models were tested to determine associations between SNPs and BPD.
In our cohort (n=659), 284 infants had BPD; 135 of whom developed severe BPD. Presence of the hypomorphic NQO1 SNP (rs1800566) in a homozygous state was associated with increased BPD while presence of the NFE2L2 SNP (rs6721961) was associated with decreased severe BPD in the entire cohort and in Caucasian infants. In regression models that adjusted for epidemiological confounders, the NQO1 and the NFE2L2 SNPs were associated with BPD and severe BPD, respectively.
Genetic variants in NFE2L2-ARE axis may contribute to the variance in liability to BPD observed in preterm infants. These results require confirmation in independent cohorts.
PMCID: PMC4522928  PMID: 25518008
7.  Exploring Concordance and Discordance for Return of Incidental Findings from Clinical Sequencing 
To explore specific conditions and types of genetic variants that specialists in genetics recommend should be returned as incidental findings in clinical sequencing.
Sixteen specialists in clinical genetics and/or molecular medicine selected variants in 99 common conditions to return to the ordering physician if discovered incidentally through whole genome sequencing. For most conditions, the specialists independently considered 3 molecular scenarios for both adults and minor children: a known pathogenic mutation, a truncating variant presumed pathogenic (where other truncating variants were known to be pathogenic), or a missense variant predicted in silico to be pathogenic.
On average, for adults and children respectively, each specialist selected 83.5 and 79.0 conditions or genes out of 99 in the known pathogenic mutation categories, 57.0 and 53.5 out of 72 in the truncating variant categories, and 33.4 and 29.7 out of 72 in the missense variant categories. Concordance in favor of disclosure within the adult/known pathogenic mutation category was 100% for 21 conditions or genes and 80% or higher for 64 conditions or genes.
Specialists were highly concordant for the return of findings in 64 conditions or genes if discovered incidentally during whole exome or whole genome sequencing.
PMCID: PMC3763716  PMID: 22422049
whole genome sequencing; incidental findings
8.  Successful immune tolerance induction to enzyme replacement therapy in CRIM-negative infantile Pompe disease 
Infantile Pompe disease resulting from a deficiency of lysosomal acid α-glucosidase (GAA) requires enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA). Cross-reactive immunologic material negative (CRIM-negative) Pompe patients develop high-titer antibody to the rhGAA and do poorly. We describe successful tolerance induction in CRIM-negative patients.
Two CRIM-negative patients with preexisting anti-GAA antibodies were treated therapeutically with rituximab, methotrexate, and gammaglobulins. Two additional CRIM-negative patients were treated prophylactically with a short course of rituximab and methotrexate, in parallel with initiating rhGAA.
In both patients treated therapeutically, anti-rhGAA was eliminated after 3 and 19 months. All four patients are immune tolerant to rhGAA, off immune therapy, showing B-cell recovery while continuing to receive ERT at ages 36 and 56 months (therapeutic) and 18 and 35 months (prophylactic). All patients show clinical response to ERT, in stark contrast to the rapid deterioration of their nontolerized CRIM-negative counterparts.
The combination of rituximab with methotrexate ± intravenous gammaglobulins (IVIG) is an option for tolerance induction of CRIM-negative Pompe to ERT when instituted in the naïve setting or following antibody development. It should be considered in other conditions in which antibody response to the therapeutic protein elicits robust antibody response that interferes with product efficacy.
PMCID: PMC3711224  PMID: 22237443
immune tolerance; methotrexate; Pompe disease; rituximab
9.  Whole-exome sequencing supports genetic heterogeneity in childhood apraxia of speech 
Childhood apraxia of speech (CAS) is a rare, severe, persistent pediatric motor speech disorder with associated deficits in sensorimotor, cognitive, language, learning and affective processes. Among other neurogenetic origins, CAS is the disorder segregating with a mutation in FOXP2 in a widely studied, multigenerational London family. We report the first whole-exome sequencing (WES) findings from a cohort of 10 unrelated participants, ages 3 to 19 years, with well-characterized CAS.
As part of a larger study of children and youth with motor speech sound disorders, 32 participants were classified as positive for CAS on the basis of a behavioral classification marker using auditory-perceptual and acoustic methods that quantify the competence, precision and stability of a speaker’s speech, prosody and voice. WES of 10 randomly selected participants was completed using the Illumina Genome Analyzer IIx Sequencing System. Image analysis, base calling, demultiplexing, read mapping, and variant calling were performed using Illumina software. Software developed in-house was used for variant annotation, prioritization and interpretation to identify those variants likely to be deleterious to neurodevelopmental substrates of speech-language development.
Among potentially deleterious variants, clinically reportable findings of interest occurred on a total of five chromosomes (Chr3, Chr6, Chr7, Chr9 and Chr17), which included six genes either strongly associated with CAS (FOXP1 and CNTNAP2) or associated with disorders with phenotypes overlapping CAS (ATP13A4, CNTNAP1, KIAA0319 and SETX). A total of 8 (80%) of the 10 participants had clinically reportable variants in one or two of the six genes, with variants in ATP13A4, KIAA0319 and CNTNAP2 being the most prevalent.
Similar to the results reported in emerging WES studies of other complex neurodevelopmental disorders, our findings from this first WES study of CAS are interpreted as support for heterogeneous genetic origins of this pediatric motor speech disorder with multiple genes, pathways and complex interactions. We also submit that our findings illustrate the potential use of WES for both gene identification and case-by-case clinical diagnostics in pediatric motor speech disorders.
PMCID: PMC3851280  PMID: 24083349
Apraxia of speech; Developmental verbal dyspraxia; Next-generation sequencing; Speech disorder; Whole-exome sequencing
12.  Correction of Hyperbilirubinemia in Gunn Rats Using Clinically Relevant Low Doses of Helper-Dependent Adenoviral Vectors 
Human Gene Therapy  2010;22(4):483-488.
Crigler–Najjar syndrome type I is a severe inborn error of bilirubin metabolism caused by a complete deficiency of uridine diphospho-glucuronosyl transferase 1A1 (UGT1A1) and results in life-threatening unconjugated hyperbilirubinemia. Lifelong correction of hyperbilirubinemia by liver-directed gene therapy using a helper-dependent adenoviral (HDAd) vector has been previously reported in the Gunn rat, a model of Crigler–Najjar syndrome, but was only achieved using high doses (≥3 × 1012 viral particles [vp]/kg), which are likely to elicit a severe toxic response in humans. Therefore, in this study, we investigate strategies to achieve correction of hyperbilirubinemia in the Gunn rat using clinically relevant low HDAd doses. We have found that correction of hyperbilirubinemia in the Gunn rat can be achieved with a low dose of 5 × 1011 vp/kg by using an HDAd vector bearing a more potent UGT1A1 expression cassette. Furthermore, by using hydrodynamic injection of the improved HDAd vector, correction of hyperbilirubinemia in the Gunn rat can be achieved using an even lower dose of 5 × 1010 vp/kg. Although hydrodynamic injection as performed in rats is not acceptable in humans, clinically attractive, minimally invasive methods have been successfully developed to mimic hydrodynamic injection of HDAd vector in non-human primates. Therefore, using an improved expression cassette combined with a more efficient method of vector delivery permits correction of hyperbilirubinemia in the Gunn rat using clinically relevant low HDAd doses and may thus pave the way to clinical application of HDAd vectors for Crigler–Najjar syndrome gene therapy.
Crigler-Najjar syndrome is an autosomal recessive inborn error of bilirubin metabolism caused by the lack of hepatic uridine diphosphoglucuronosyltransferase 1A1 (UGT1A1) activity. In this study, Dimmock and colleagues demonstrate that hydrodynamic injection of a helper-dependent adenoviral (HDAd) vector encoding UGT1A1 leads to correction of hyperbilirubinemia in the Gunn at doses as low as 5 × 1010 VP/kg.
PMCID: PMC3073075  PMID: 20973621
13.  The Role of Molecular Testing and Enzyme Analysis in the Management of Hypomorphic Citrullinemia 
Expanded newborn screening detects patients with modest elevations in citrulline; however it is currently unclear how to treat these patients and how to counsel their parents. In order to begin to address these issues, we compared the clinical, biochemical and molecular features of 10 patients with mildly elevated citrulline levels. Three patients presented with clinical illness whereas seven came to attention as a result of expanded newborn screening. One patient presented during pregnancy and responded promptly to IV sodium phenylacetate/sodium benzoate and arginine therapy with no long-term adverse effects on mother or fetus. Two children presented with neurocognitive dysfunction, one of these responded dramatically to dietary protein reduction. ASS enzyme activity was not deficient in all patients with biallelic mutations suggesting this test cannot exclude the ASS1 locus in patients with mildly elevated plasma citrulline. Conversely, all symptomatic patients who were tested had deficient activity. We describe four unreported mutations (p.Y291S, p.R272H, p.F72L and p.L88I), as well as the common p.W179R mutation. In Silico algorithms were inconsistent in predicting the pathogenicity of mutations. The cognitive benefit in one patient of protein restriction and the lack of adverse outcome in 7 others restricted from birth, suggest a role for protein restriction and continued monitoring to prevent neurocognitive dysfunction.
PMCID: PMC2597641  PMID: 18925679
ASS1; Liver Failure; Drug Therapy; Pregnancy; DNA Diagnosis; Newborn Screening

Results 1-13 (13)