Improving the diagnosis of cobalamin and related defects by genomic analysis, plus functional and structural assessment of novel variants.

BACKGROUND: Cellular cobalamin defects are a locus and allelic heterogeneous disorder. The gold standard for coming to genetic diagnoses of cobalamin defects has for some time been gene-by-gene Sanger sequencing of individual DNA fragments. Enzymatic and cellular methods are employed before such sequencing to help in the selection of the gene defects to be sought, but this is time-consuming and laborious. Furthermore some cases remain undiagnosed because no biochemical methods have been available to test for cobalamin absorption and transport defects. RESULTS: This paper reports the use of massive parallel sequencing of DNA (exome analysis) for the accurate and rapid genetic diagnosis of cobalamin-related defects in a cohort of affected patients. The method was first validated in an initial cohort with different cobalamin defects. Mendelian segregation, the frequency of mutations, and the comprehensive structural and functional analysis of gene variants, identified disease-causing mutations in 12 genes involved in the absorption and synthesis of active cofactors of vitamin B12 (22 cases), and in the non-cobalamin metabolism-related genes ACSF3 (in four biochemically misdiagnosed patients) and SUCLA2 (in one patient with an unusual presentation). We have identified thirteen new variants all classified as pathogenic according to the ACGM recommendation but four were classified as variant likely pathogenic in MUT and SUCLA2. Functional and structural analysis provided evidences to classify them as pathogenic variants. CONCLUSIONS: The present findings suggest that the technology used is sufficiently sensitive and specific, and the results it provides sufficiently reproducible, to recommend its use as a second-tier test after the biochemical detection of cobalamin disorder markers in the first days of life. However, for accurate diagnoses to be made, biochemical and functional tests that allow comprehensive clinical phenotyping are also needed.

Nonketotic hyperglycinemia: Functional assessment of missense variants in GLDC to understand phenotypes of the disease.

The rapid analysis of genomic data is providing effective mutational confirmation in patients with clinical and biochemical hallmarks of a specific disease. This is the case for nonketotic hyperglycinemia (NKH), a Mendelian disorder causing seizures in neonates and early-infants, primarily due to mutations in the GLDC gene. However, understanding the impact of missense variants identified in this gene is a major challenge for the application of genomics into clinical practice. Herein, a comprehensive functional and structural analysis of 19 GLDC missense variants identified in a cohort of 26 NKH patients was performed. Mutant cDNA constructs were expressed in COS7 cells followed by enzymatic assays and Western blot analysis of the GCS P-protein to assess the residual activity and mutant protein stability. Structural analysis, based on molecular modeling of the 3D structure of GCS P-protein, was also performed. We identify hypomorphic variants that produce attenuated phenotypes with improved prognosis of the disease. Structural analysis allows us to interpret the effects of mutations on protein stability and catalytic activity, providing molecular evidence for clinical outcome and disease severity. Moreover, we identify an important number of mutants whose loss-of-functionality is associated with instability and, thus, are potential targets for rescue using folding therapeutic approaches.

Longitudinal volumetric and 2D assessment of cerebellar atrophy in a large cohort of children with phosphomannomutase deficiency (PMM2-CDG).

OBJECTIVE: We aim to delineate the progression of cerebellar atrophy (the primary neuroimaging finding) in children with phosphomannomutase-deficiency (PMM2-CDG) by analyzing longitudinal MRI studies and performing cerebellar volumetric analysis and a 2D cerebellar measurement. METHODS: Statistical analysis was used to compare MRI measurements [midsagittal vermis relative diameter (MVRD) and volume] of children with PMM2-CDG and sex- and age-matched controls, and to determine the rate of progression of cerebellar atrophy at different ages. RESULTS: Fifty MRI studies of 33 PMM2-CDG patients were used for 2D evaluation, and 19 MRI studies were available for volumetric analysis. Results from a linear regression model showed that patients have a significantly lower MVRD and cerebellar volume compared to controls (p < 0.001 and p < 0.001 respectively). There was a significant negative correlation between age and MVRD for patients (p = 0.014). The rate of cerebellar atrophy measured by the loss of MVRD and cerebellar volume per year was higher at early ages (r = -0.578, p = 0.012 and r = -0.323, p = 0.48 respectively), particularly in patients under 11 years (p = 0.004). There was a significant positive correlation between MVRD and cerebellar volume in PMM2-CDG patients (r = 0.669, p = 0.001). CONCLUSIONS: Our study quantifies a progression of cerebellar atrophy in PMM2-CDG patients, particularly during the first decade of life, and suggests a simple and reliable measure, the MVRD, to monitor cerebellar atrophy. Quantitative measurement of MVRD and cerebellar volume are essential for correlation with phenotype and outcome, natural follow-up, and monitoring in view of potential therapies in children with PMM2-CDG.

Phosphomannomutase deficiency (PMM2-CDG): ataxia and cerebellar assessment.

BACKGROUND: Phosphomannomutase deficiency (PMM2-CDG) is the most frequent congenital disorder of glycosylation. The cerebellum is nearly always affected in PMM2-CDG patients, a cerebellar atrophy progression is observed, and cerebellar dysfunction is their main daily functional limitation. Different therapeutic agents are under development, and clinical evaluation of drug candidates will require a standardized score of cerebellar dysfunction. We aim to assess the validity of the International Cooperative Ataxia Rating Scale (ICARS) in children and adolescents with genetically confirmed PMM2-CDG deficiency. We compare ICARS results with the Nijmegen Pediatric CDG Rating Scale (NPCRS), neuroimaging, intelligence quotient (IQ) and molecular data. METHODS: Our observational study included 13 PMM2-CDG patients and 21 control subjects. Ethical permissions and informed consents were obtained. Three independent child neurologists rated PMM2-CDG patients and control subjects using the ICARS. A single clinician administered the NPCRS. All patients underwent brain MRI, and the relative diameter of the midsagittal vermis was measured. Psychometric evaluations were available in six patients. The Mann-Whitney U test was used to compare ICARS between patients and controls. To evaluate inter-observer agreement in patients' ICARS ratings, intraclass correlation coefficients (ICC) were calculated. ICARS internal consistency was evaluated using Cronbach's alpha. Spearman's rank correlation coefficient test was used to correlate ICARS with NPCRS, midsagittal vermis relative diameter and IQ. RESULTS: ICARS and ICARS subscores differed between patients and controls (p < 0.001). Interobserver agreement of ICARS was "almost perfect" (ICC = 0.99), with a "good" internal reliability (Cronbach's alpha = 0.72). ICARS was significantly correlated with the total NPCRS score (rs 0.90, p < 0.001). However, there was no agreement regarding categories of severity. Regarding neuroimaging, inverse correlations between ICARS and midsagittal vermis relative diameter (rs -0.85, p = 0.003) and IQ (rs -0.94, p = 0.005) were found. Patients bearing p.E93A, p.C241S or p.R162W mutations presented a milder phenotype. CONCLUSIONS: ICARS is a reliable instrument for assessment of PMM2-CDG patients, without significant inter-rater variability. Despite our limited sample size, the results show a good correlation between functional cerebellar assessment, IQ and neuroimaging. For the first a correlation between ICARS, neuroimaging and IQ in PMM2-CDG patients has been demonstrated.

Cost-Effectiveness Analysis of a National Newborn Screening Program for Biotinidase Deficiency.

BACKGROUND AND OBJECTIVES: There are conflicting views as to whether testing for biotinidase deficiency (BD) ought to be incorporated into universal newborn screening (NBS) programs. The aim of this study was to evaluate the cost-effectiveness of adding BD to the panel of conditions currently screened under the national NBS program in Spain. METHODS: We used information from the regional NBS program for BD that has been in place in the Spanish region of Galicia since 1987. These data, along with other sources, were used to develop a cost-effectiveness decision model that compared lifetime costs and health outcomes of a national birth cohort of newborns with and without an early detection program. The analysis took the perspective of the Spanish National Health Service. Effectiveness was measured in terms of quality-adjusted life years (QALYs). We undertook extensive sensitivity analyses around the main model assumptions, including a probabilistic sensitivity analysis. RESULTS: In the base case analysis, NBS for BD led to higher QALYs and higher health care costs, with an estimated incremental cost per QALY gained of $24,677. Lower costs per QALY gained were found when conservative assumptions were relaxed, yielding cost savings in some scenarios. The probability that BD screening was cost-effective was estimated to be >70% in the base case at a standard threshold value. CONCLUSIONS: This study indicates that NBS for BD is likely to be a cost-effective use of resources.