Red Blood Cell Metabolism in Pyruvate Kinase Deficient Patients.

Background: Pyruvate kinase deficiency (PKD) is the most frequent congenital enzymatic defect of glycolysis, and one of the most common causes of hereditary non spherocytic hemolytic anemia. Therapeutic interventions are limited, in part because of the incomplete understanding of the molecular mechanisms that compensate for the metabolic defect. Methods: Mass spectrometry-based metabolomics analyses were performed on red blood cells (RBCs) from healthy controls (n=10) and PKD patients (n=5). Results: In PKD patients, decreases in late glycolysis were accompanied by accumulation of pentose phosphate pathway (PPP) metabolites, as a function of oxidant stress to purines (increased breakdown and deamination). Markers of oxidant stress included increased levels of sulfur-containing compounds (methionine and taurine), polyamines (spermidine and spermine). Markers of hypoxia such as succinate, sphingosine 1-phosphate (S1P), and hypoxanthine were all elevated in PKD subjects. Membrane lipid oxidation and remodeling was observed in RBCs from PKD patients, as determined by increases in the levels of free (poly-/highly-unsaturated) fatty acids and acyl-carnitines. Conclusion: In conclusion, in the present study, we provide the first overview of RBC metabolism in patients with PKD. Though limited in scope, the study addresses the need for basic science to investigate pathologies targeting underrepresented minorities (Amish population in this study), with the ultimate goal to target treatments to health disparities.

Genotype-phenotype correlation and molecular heterogeneity in pyruvate kinase deficiency.

Pyruvate kinase (PK) deficiency is a rare recessive congenital hemolytic anemia caused by mutations in the PKLR gene. This study reports the molecular features of 257 patients enrolled in the PKD Natural History Study. Of the 127 different pathogenic variants detected, 84 were missense and 43 non-missense, including 20 stop-gain, 11 affecting splicing, five large deletions, four in-frame indels, and three promoter variants. Within the 177 unrelated patients, 35 were homozygous and 142 compound heterozygous (77 for two missense, 48 for one missense and one non-missense, and 17 for two non-missense variants); the two most frequent mutations were p.R510Q in 23% and p.R486W in 9% of mutated alleles. Fifty-five (21%) patients were found to have at least one previously unreported variant with 45 newly described mutations. Patients with two non-missense mutations had lower hemoglobin levels, higher numbers of lifetime transfusions, and higher rates of complications including iron overload, extramedullary hematopoiesis, and pulmonary hypertension. Rare severe complications, including lower extremity ulcerations and hepatic failure, were seen more frequently in patients with non-missense mutations or with missense mutations characterized by severe protein instability. The PKLR genotype did not correlate with the frequency of complications in utero or in the newborn period. With ICCs ranging from 0.4 to 0.61, about the same degree of clinical similarity exists within siblings as it does between siblings, in terms of hemoglobin, total bilirubin, splenectomy status, and cholecystectomy status. Pregnancy outcomes were similar across genotypes in PK deficient women. This report confirms the wide genetic heterogeneity of PK deficiency.

A critical reappraisal of dietary practices in methylmalonic acidemia raises concerns about the safety of medical foods. Part 2: cobalamin C deficiency.

PURPOSE: Cobalamin C (cblC) deficiency impairs the biosynthesis of 5'-deoxyadenosyl-adenosyl- and methyl-cobalamin, resulting in methylmalonic acidemia combined with hyperhomocysteinemia and hypomethioninemia. However, some patients with cblC deficiency are treated with medical foods, devoid of methionine and high in leucine content, that are formulated for patients with isolated propionate oxidative defects. We examined the effects of imbalanced branched-chain amino acid intake on growth outcomes in cblC-deficient patients. METHODS: Dietary intake was correlated with biochemical, anthropometric, and body composition measurements and other disease parameters in a cohort of 28 patients with early-onset cblC deficiency. RESULTS: Protein-restricted diets were followed by 21% of the patients, whereas 32% received medical foods. Patients on protein-restricted diets had lower height-for-age z-score (P = 0.034), whereas patients consuming medical foods had lower head circumference Z-scores (P = 0.037), plasma methionine concentrations (P = 0.001), and predicted methionine influx through the blood-brain barrier Z-score (-1.29 vs. -0.0617; P = 0.007). The combination of age at diagnosis, a history of seizures, and the leucine-to-valine dietary intake ratio best predicted head circumference Z-score based on multiple regression modeling (R(2) = 0.945). CONCLUSIONS: Patients with cblC deficiency treated with medical foods designed for isolated methylmalonic acidemia are at risk for iatrogenic methionine deficiency that could adversely affect brain growth and development.Genet Med 18 4, 396-404.

Development of DNA confirmatory and high-risk diagnostic testing for newborns using targeted next-generation DNA sequencing.

PURPOSE: Genetic testing is routinely used for second-tier confirmation of newborn sequencing results to rule out false positives and to confirm diagnoses in newborns undergoing inpatient and outpatient care. We developed a targeted next-generation sequencing panel coupled with a variant processing pipeline and demonstrated utility and performance benchmarks across multiple newborn disease presentations in a retrospective clinical study. METHODS: The test utilizes an in silico gene filter that focuses directly on 126 genes related to newborn screening diseases and is applied to the exome or a next-generation sequencing panel called NBDx. NBDx targets the 126 genes and additional newborn-specific disorders. It integrates DNA isolation from minimally invasive biological specimens, targeted next-generation screening, and rapid characterization of genetic variation. RESULTS: We report a rapid parallel processing of 8 to 20 cases within 105 hours with high coverage on our NBDx panel. Analytical sensitivity of 99.8% was observed across known mutation hotspots. Concordance calls with or without clinical summaries were 94% and 75%, respectively. CONCLUSION: Rapid, automated targeted next-generation sequencing and analysis are practical in newborns for second-tier confirmation and neonatal intensive care unit diagnoses, laying a foundation for future primary DNA-based molecular screening of additional disorders and improving existing molecular testing options for newborns.

A homozygous SLITRK6 nonsense mutation is associated with progressive auditory neuropathy in humans.

OBJECTIVES/HYPOTHESIS: SLITRK family proteins control neurite outgrowth and regulate synaptic development. In mice, Slitrk6 plays a role in the survival and innervation of sensory neurons in the inner ear, vestibular apparatus, and retina, and also influences axial eye length. We provide the first detailed description of the auditory phenotype in humans with recessive SLITRK6 deficiency. STUDY DESIGN: Prospective observational case study. METHODS: Nine closely related Amish subjects from an endogamous Amish community of Pennsylvania underwent audiologic and vestibular testing. Single nucleotide polymorphism microarrays were used to map the chromosome locus, and Sanger sequencing or high-resolution melt analysis were used to confirm the allelic variant. RESULTS: All nine subjects were homozygous for a novel nonsense variant of SLITRK6 (c.1240C>T, p.Gln414Ter). Adult patients had high myopia. The 4 oldest SLITRK6 c.1240C>T homozygotes had absent ipsilateral middle ear muscle reflexes (MEMRs). Distortion product otoacoustic emissions (DPOAEs) were absent in all ears tested and the cochlear microphonic (CM) was increased in amplitude and duration in young patients and absent in the two oldest subjects. Auditory brainstem responses (ABRs) were dys-synchronised bilaterally with no reproducible waves I, III, or V at high intensities. Hearing loss and speech reception thresholds deteriorated symmetrically with age, which resulted in severe-to-profound hearing impairment by early adulthood. Vestibular evoked myogenic potentials were normal in three ears and absent in one. CONCLUSION: Homozygous SLITRK6 c.1240C>T (p.Gln414Ter) nonsense mutations are associated with high myopia, cochlear dysfunction attributed to outer hair cell disease, and progressive auditory neuropathy.

Identification of a sporozoite-specific antigen from Toxoplasma gondii.

Reduction of risk for human and food animal infection with Toxoplasma gondii is hampered by the lack of epidemiological data documenting the predominant routes of infection (oocyst vs. tissue cyst consumption) in horizontally transmitted toxoplasmosis. Existing serological assays can determine previous exposure to the parasite, but not the route of infection. We have used difference gel electrophoresis, in combination with tandem mass spectroscopy and Western blot, to identify a sporozoite-specific protein (T. gondii embryogenesis-related protein [TgERP]), which elicited antibody and differentiated oocyst- versus tissue cyst-induced infection in pigs and mice. The recombinant protein was selected from a cDNA library constructed from T. gondii sporozoites; this protein was used in Western blots and probed with sera from T. gondii -infected humans. Serum antibody to TgERP was detected in humans within 6-8 mo of initial oocyst-acquired infection. Of 163 individuals in the acute stage of infection (anti- T. gondii IgM detected in sera, or < 30 in the IgG avidity test), 103 (63.2%) had detectable antibodies that reacted with TgERP. Of 176 individuals with unknown infection route and in the chronic stage of infection (no anti- T. gondii IgM detected in sera, or > 30 in the IgG avidity test), antibody to TgERP was detected in 31 (17.6%). None of the 132 uninfected individuals tested had detectable antibody to TgERP. These data suggest that TgERP may be useful in detecting exposure to sporozoites in early T. gondii infection and implicates oocysts as the agent of infection.

Osteoporosis-pseudoglioma syndrome: description of 9 new cases and beneficial response to bisphosphonates.

Osteoporosis-pseudoglioma syndrome (OPPG) is a rare autosomal recessive disorder of severe juvenile osteoporosis and congenital blindness, due to mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene. Approximately fifty cases of OPPG have been reported. We report 9 new cases of OPPG, in three related nuclear families of Conservative Mennonites in Pennsylvania. All 9 children with OPPG were blind and had osteoporosis. Four of six parents had low bone mineral density (BMD) or osteoporosis; 2 were normal. Sequence analysis from genomic DNA revealed homozygosity for a nonsense mutation of exon 6 of LRP5 (W425X) in four OPPG cases tested in families A and C. In family B, OPPG cases were compound heterozygotes for the exon 6 W425X LRP5 mutation and a second exon 6 mutation (T409A); bone phenotype was milder than in family A. Neither of these mutations was present in an unrelated normal. The four treated OPPG patients all responded to bisphosphonates (duration 1.5-6.5 years) with improvement in Z-scores. One patient had a negligible response to teriparatide. In summary, we report 9 new cases of OPPG due to two novel LRP5 mutations, note a milder bone phenotype but similar ocular phenotype in LRP5 W425X/T409A compound heterozygotes than in W425X homozygotes and describe positive response to bisphosphonate treatment in four cases.