Research Spotlight

Choquet, K., D. Forget, E. Meloche, M. J. Dicaire, G. Bernard, A. Vanderver, R. Schiffmann, M. R. Fabian, M. Teichmann, B. Coulombe, B. Brais and C. L. Kleinman (2019). “Leukodystrophy-associated POLR3A mutations down-regulate the RNA polymerase III transcript and important regulatory RNA BC200.” J Biol Chem 294(18): 7445-7459.

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RNA polymerase III (Pol III) is an essential enzyme responsible for the synthesis of several small noncoding RNAs, a number of which are involved in mRNA translation. Recessive mutations in POLR3A, encoding the largest subunit of Pol III, cause POLR3-related hypomyelinating leukodystrophy (POLR3-HLD), characterized by deficient central nervous system myelination. Identification of the downstream effectors of pathogenic POLR3A mutations has so far been elusive. Here, we used CRISPR-Cas9 to introduce the POLR3A mutation c.2554A–>G (p.M852V) into human cell lines and assessed its impact on Pol III biogenesis, nuclear import, DNA occupancy, transcription, and protein levels. Transcriptomic profiling uncovered a subset of transcripts vulnerable to Pol III hypofunction, including a global reduction in tRNA levels. The brain cytoplasmic BC200 RNA (BCYRN1), involved in translation regulation, was consistently affected in all our cellular models, including patient-derived fibroblasts. Genomic BC200 deletion in an oligodendroglial cell line led to major transcriptomic and proteomic changes, having a larger impact than those of POLR3A mutations. Upon differentiation, mRNA levels of the MBP gene, encoding myelin basic protein, were significantly decreased in POLR3A-mutant cells. Our findings provide the first evidence for impaired Pol III transcription in cellular models of POLR3-HLD and identify several candidate effectors, including BC200 RNA, having a potential role in oligodendrocyte biology and involvement in the disease.

Brown, M. N., D. C. Walters, M. A. Schmidt, J. Hill, A. McConnell, E. Jansen, G. S. Salomons, E. Arning, T. Bottiglieri, K. M. Gibson and J. B. Roullet (2019). “Maternal Glutamine Supplementation in Murine Succinic Semialdehyde Dehydrogenase Deficiency (SSADHD), a Disorder of GABA Metabolism.” J Inherit Metab Dis Apr 29. [Epub ahead of print].

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Murine succinic semialdehyde dehydrogenase deficiency (SSADHD) manifests with high concentrations of gamma-aminobutyric acid (GABA) and gamma-hydroxybutyrate (GHB) and low glutamine in the brain. To understand the pathogenic contribution of central glutamine deficiency, we exposed aldh5a1(-/-) (SSADHD) mice and their genetic controls (aldh5a1(+/+) ) to either a 4% (w/w) glutamine-containing diet or a glutamine-free diet from conception until post-natal day 30. Endpoints included brain, liver and blood amino acids, brain GHB, ataxia scores and open field testing. Glutamine supplementation did not improve aldh5a1(-/-) brain glutamine deficiency nor brain GABA and GHB. It decreased brain glutamate but did not change the ratio of excitatory (glutamate) to inhibitory (GABA) neurotransmitters. In contrast, glutamine supplementation significantly increased brain arginine (30% for aldh5a1(+/+) and 18% for aldh5a1(-/-) mice), and leucine (12% and 18%). Glutamine deficiency was confirmed in the liver. The test diet increased hepatic glutamate in both genotypes, decreased glutamine in aldh5a1(+/+) but not in aldh5a1(-/-) , but had no effect on GABA. Dried bloodspot analyses showed significantly elevated GABA in mutants (~800% above controls) and decreased glutamate (~25%), but no glutamine difference with controls. Glutamine supplementation did not impact blood GABA but significantly increased glutamine and glutamate in both genotypes indicating systemic exposure to dietary glutamine. Ataxia and pronounced hyperactivity were observed in aldh5a1(-/-) mice but remained unchanged by the diet intervention. The study suggests that glutamine supplementation improves peripheral but not central glutamine deficiency in experimental SSADHD. Future studies are needed to fully understand the pathogenic role of brain glutamine deficiency in SSADHD.

Brown, M., P. Ashcraft, E. Arning, T. Bottiglieri, W. McClintock, F. Giancola, D. Lieberman, N. S. Hauser, R. Miller, J. B. Roullet, P. Pearl and K. M. Gibson (2019). “Rett syndrome (MECP2) and succinic semialdehyde dehydrogenase (ALDH5A1) deficiency in a developmentally delayed female.” Mol Genet Genomic Med 7(5): e629.

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BACKGROUND: We present a patient with Rett syndrome (RTT; MECP2) and autosomal-recessive succinic semialdehyde dehydrogenase deficiency (SSADHD; ALDH5A1 (aldehyde dehydrogenase 5a1 = SSADH), in whom the current phenotype exhibits features of SSADHD (hypotonia, global developmental delay) and RTT (hand stereotypies, gait anomalies). METHODS: gamma-Hydroxybutyric acid (GHB) was quantified by UPLC-tandem mass spectrometry, while mutation analysis followed standard methodology of whole-exome sequencing. RESULTS: The biochemical hallmark of SSADHD, GHB was increased in the proband’s dried bloodspot (DBS; 673 microM; previous SSADHD DBSs (n = 7), range 124-4851 microM); control range (n = 2,831), 0-78 microM. The proband was compound heterozygous for pathogenic ALDH5A1 mutations (p.(Asn418IlefsTer39); maternal; p.(Gly409Asp); paternal) and a de novo RTT nonsense mutation in MECP2 (p.Arg255*). CONCLUSION: The major inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), is increased in SSADHD but normal in RTT, although there are likely regional changes in GABA receptor distribution. GABAergic anomalies occur in both disorders, each featuring an autism spectrum phenotype. What effect the SSADHD biochemical anomalies (elevated GABA, GHB) might play in the neurodevelopmental/epileptic phenotype of our patient is currently unknown.

Bishop, M. E., S. R. Black, J. Nguyen, D. Mintz and B. S. Stein (2019). “A Simple Method of Measuring the Distance From the Schottle Point to the Medial Distal Femoral Physis With MRI.” Orthop J Sports Med 7(4): eCollection.

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Background: Medial patellofemoral ligament (MPFL) reconstruction is the treatment of choice for recurrent patellar instability in the skeletally immature patient. Avoiding the open physes during anatomic MPFL reconstruction is a challenge in this population. Purpose: To describe a novel method using magnetic resonance imaging (MRI) to determine the distance from the Schottle point to the medial distal femoral physis among skeletally immature individuals with patellar instability. Study Design: Descriptive laboratory study. Methods: Preoperative MRI scans were analyzed from 34 patients with open distal femoral physes and lateral patellar instability. With the multiplanar reconstruction mode on a picture archiving and communication system (PACS), the location of the Schottle point was determined according to previously reported distances from the posterior femoral cortical line and the posterior origin of the medial femoral condyle. This location was then extrapolated to the most medial sagittal slice on MRI showing the medial distal femoral physis. The distance was measured from this point to the most distal aspect of the physis. Results: The mean age of the study cohort was 13.6 years (range, 10.6-15.7 years); there were 13 males and 21 females. The mean distance from the medial distal femoral physis to the Schottle point was 7.27 +/- 1.78 mm. The Schottle point was distal to the medial distal femoral physis in all cases. There was no significant correlation between age and mean distance in either the overall study population (r = 0.046, P = .798) or when stratified by sex (females, P = .629; males, P = .089). The distance between the Schottle point and the medial distal femoral physis was shorter for females than for males (6.51 vs 7.71 mm, P = .043). After adjustment for age, females on average were 1.31 mm closer to the Schottle point than were males (B = -1.31, P = .041). Conclusion: This technique can be used to determine the distance between the medial distal femoral physis and the Schottle point. The Schottle point was distal to the physis in all patients, and it was closer to the physis in skeletally immature females compared with age-matched males. Clinical Relevance: The long-term repercussions of improperly placed MPFL reconstruction include recurrent patellar instability, increased patellofemoral contact pressures and overtensioning of the ligament, and possibly patellofemoral arthritis. The current technique can be used preoperatively to determine the appropriate safe distance for drilling a socket distal to the physis.

Balmforth, C., J. Simpson, L. Shen, P. S. Jhund, M. Lefkowitz, A. R. Rizkala, J. L. Rouleau, V. Shi, S. D. Solomon, K. Swedberg, M. R. Zile, M. Packer and J. J. V. McMurray (2019). “Outcomes and Effect of Treatment According to Etiology in HFrEF: An Analysis of PARADIGM-HF.” JACC Heart Fail May 3. [Epub ahead of print].

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OBJECTIVES: The purpose of this study was to compare outcomes (and the effect of sacubitril/valsartan) according to etiology in the PARADIGM-HF (Prospective comparison of angiotensin-receptor-neprilysin inhibitor [ARNI] with angiotensin-converting-enzyme inhibitor [ACEI] to Determine Impact on Global Mortality and morbidity in Heart Failure) trial. BACKGROUND: Etiology of heart failure (HF) has changed over time in more developed countries and is also evolving in non-Western societies. Outcomes may vary according to etiology, as may the effects of therapy. METHODS: We examined outcomes and the effect of sacubtril/valsartan according to investigator-reported etiology in PARADIGM-HF. The outcomes analyzed were the primary composite of cardiovascular death or HF hospitalization, and components, and death from any cause. Outcomes were adjusted for known prognostic variables including N terminal pro-B type natriuretic peptide. RESULTS: Among the 8,399 patients randomized, 5,036 patients (60.0%) had an ischemic etiology. Among the 3,363 patients (40.0%) with a nonischemic etiology, 1,595 (19.0% of all patients; 47% of nonischemic patients) had idiopathic dilated cardiomyopathy, 968 (11.5% of all patients; 28.8% of nonischemic patients) had a hypertensive cause, and 800 (9.5% of all patients, 23.8% of nonischemic patients) another cause (185 infective/viral, 158 alcoholic, 110 valvular, 66 diabetes, 30 drug-related, 14 peripartum-related, and 237 other). Whereas the unadjusted rates of all outcomes were highest in patients with an ischemic etiology, the adjusted hazard ratios (HRs) were not different from patients in the 2 major nonischemic etiology categories; for example, for the primary outcome, compared with ischemic (HR: 1.00), hypertensive 0.87 (95% confidence interval [CI]: 0.75 to 1.02), idiopathic 0.92 (95% CI: 0.82 to 1.04) and other 1.00 (95% CI: 0.85 to 1.17). The benefit of sacubitril/valsartan over enalapril was consistent across etiologic categories (interaction for primary outcome; p = 0.11). CONCLUSIONS: Just under half of patients in this global trial had nonischemic HF with reduced ejection fraction, with idiopathic and hypertensive the most commonly ascribed etiologies. Adjusted outcomes were similar across etiologic categories, as was the benefit of sacubitril/valsartan over enalapril. (Efficacy and Safety of LCZ696 Compared to Enalapril on Morbidity and Mortality of Patients With Chronic Heart Failure; NCT01035255).