PattRec: An easy-to-use CNV detection tool optimized for targeted NGS assays with diagnostic purposes.

Genetic laboratories use custom-commercial targeted next-generation sequencing (tg-NGS) assays to identify disease-causing variants. Although the high coverage achieved with these tests allows for the detection of copy number variants (CNVs), which account for an important proportion of the genetic burden in human diseases, an easy-to-use tool for automatic CNV detection is still lacking. This article presents a new CNV detection tool optimized for tg-NGS data: PattRec. PattRec was evaluated using a wide range of data, and its performance compared with those of other CNV detection tools. The software includes features for selecting optimal controls, discarding polymorphic CNVs prior to analysis, and filtering out deletions based on SNV zygosity, and automatically creates an in-house CNV database. There is no need for high level bioinformatic expertise and users can choose color-coded xlsx output that helps to prioritize potentially pathogenic CNVs. PattRec is presented as a Java based GUI, freely available online: https://github.com/irotero/PattRec.

Rare Variants in 48 Genes Account for 42% of Cases of Epilepsy With or Without Neurodevelopmental Delay in 246 Pediatric Patients.

In order to characterize the genetic architecture of epilepsy in a pediatric population from the Iberian Peninsula (including the Canary Islands), we conducted targeted exome sequencing of 246 patients with infantile-onset seizures with or without neurodevelopmental delay. We detected 107 variants in 48 different genes, which were implicated in neuronal excitability, neurodevelopment, synaptic transmission, and metabolic pathways. In 104 cases (42%) we detected variant(s) that we classified as pathogenic or likely pathogenic. Of the 48 mutated genes, 32 were dominant, 8 recessive and 8 X-linked. Of the patients for whom family studies could be performed and in whom pathogenic variants were identified in dominant or X-linked genes, 82% carried de novo mutations. The involvement of small copy number variations (CNVs) is 9%. The use of progressively updated custom panels with high mean vertical coverage enabled establishment of a definitive diagnosis in a large proportion of cases (42%) and detection of CNVs (even duplications) with high fidelity. In 10.5% of patients we detected associations that are pending confirmation via functional and/or familial studies. Our findings had important consequences for the clinical management of the probands, since a large proportion of the cohort had been clinically misdiagnosed, and their families were subsequently able to avail of genetic counseling. In some cases, a more appropriate treatment was selected for the patient in question, or an inappropriate treatment discontinued. Our findings suggest the existence of modifier genes that may explain the incomplete penetrance of some epilepsy-related genes. We discuss possible reasons for non-diagnosis and future research directions. Further studies will be required to uncover the roles of structural variants, epimutations, and oligogenic inheritance in epilepsy, thereby providing a more complete molecular picture of this disease. In summary, given the broad phenotypic spectrum of most epilepsy-related genes, efficient genomic tools like the targeted exome sequencing panel described here are essential for early diagnosis and treatment, and should be implemented as first-tier diagnostic tools for children with epilepsy without a clear etiologic basis.

Evolution of tyrosinemia type 1 disease in patients treated with nitisinone in Spain.

Treatment with nitisinone (NTBC) has brought about a drastic improvement in the treatment and prognosis of hereditary tyrosinemia type I (HT1). We conducted a retrospective observational multicentric study in Spanish HT1 patients treated with NTBC to assess clinical and biochemical long-term evolution.We evaluated 52 patients, 7 adults and 45 children, treated with NTBC considering: age at diagnosis, diagnosis by clinical symptoms, or by newborn screening (NBS); phenotype (acute/subacute/chronic), mutational analysis; symptoms at diagnosis and clinical course; biochemical markers; doses of NTBC; treatment adherence; anthropometric evolution; and neurocognitive outcome.The average follow-up period was 6.1 ±â€Š4.9 and 10.6 ±â€Š5.4 years in patients with early and late diagnosis respectively. All patients received NTBC from diagnosis with an average dose of 0.82 mg/kg/d. All NBS-patients (n = 8) were asymptomatic at diagnosis except 1 case with acute liver failure, and all remain free of liver and renal disease in follow-up. Liver and renal affectation was markedly more frequent at diagnosis in patients with late diagnosis (P < .001 and .03, respectively), with ulterior positive hepatic and renal course in 86.4% and 93.2% of no-NBS patients, although 1 patient with good metabolic control developed hepatocarcinoma.Despite a satisfactory global nutritional evolution, 46.1% of patients showed overweight/obesity. Interestingly lower body mass index was observed in patients with good dietary adherence (20.40 ±â€Š4.43 vs 24.30 ±â€Š6.10; P = .08) and those with good pharmacological adherence (21.19 ±â€Š4.68 vs 28.58 ±â€Š213.79).intellectual quotient was ≥85 in all NBS- and 68.75% of late diagnosis cases evaluated, 15% of which need pedagogical support, and 6.8% (3/44) showed school failure.Among the 12 variants identified in fumarylacetoacetate hydrolase gene, 1 of them novel (H63D), the most prevalent in Spanish population is c.554-1 G>T.After NTBC treatment a reduction in tyrosine and alpha-fetoprotein levels was observed in all the study groups, significant for alpha-fetoprotein in no NBS-group (P = .03), especially in subacute/chronic forms (P = .018).This series confirms that NTBC treatment had clearly improved the prognosis and quality of life of HT1 patients, but it also shows frequent cognitive dysfunctions and learning difficulties in medium-term follow-up, and, in a novel way, a high percentage of overweight/obesity.

Free-access copy-number variant detection tools for targeted next-generation sequencing data.

Copy number variants (CNVs) are intermediate-scale structural variants containing copy number changes involving DNA fragments of between 1 kb and 5 Mb. Although known to account for a significant proportion of the genetic burden in human disease, the role of CNVs (especially small CNVs) is often underestimated, as they are undetectable by traditional Sanger sequencing. Since the development of next-generation sequencing (NGS) technologies, several research groups have compared depth of coverage (DoC) patterns between samples, an approach that may facilitate effective CNV detection. Most CNV detection tools based on DoC comparisons are designed to work with whole-genome sequencing (WGS) or whole-exome sequencing (WES) data. However, few methods developed to date are designed for custom/commercial targeted NGS (tg-NGS) panels, the assays most commonly used for diagnostic purposes. Moreover, the development and evaluation of these tools is hindered by (i) the scarcity of thoroughly annotated data containing CNVs and (ii) a dearth of simulation tools for WES and tg-NGS that mimic the errors and biases encountered in these data. Here, we review DoC-based CNV detection methods described in the current literature, assess their performance with simulated tg-NGS data, and discuss their strengths and weaknesses when integrated into the daily laboratory workflow. Our findings suggest that the best methods for CNV detection in tg-NGS panels are DECoN, ExomeDepth, and ExomeCNV. Regardless of the method used, there is a need to make these programs more user-friendly to enable their use by diagnostic laboratory staff who lack bioinformatics training.

Carbohydrate status in patients with phenylketonuria.

BACKGROUND: In patients with phenylketonuria (PKU), a low-phenylalanine (Phe) diet supplemented with low-protein foods and a Phe-free amino acid mixture favors a dietary intake rich in carbohydrates, but little is known about how these molecules are metabolized in this setting. The objective of the present study was to analyze carbohydrate metabolism in patients with hyperphenylalaninemia. METHODS: We conducted a multicenter cross-sectional study to investigate biochemical markers of basal and postprandial carbohydrate metabolism in PKU patients according to age, Phe tolerance, waist circumference and body mass index (BMI), diet, tetrahydrobiopterin (BH4) supplementation, and adherence to treatment. Basal biomarkers and anthropometric parameters were also evaluated in patients with mild hyperphenylalaninemia (MHPA) and in healthy controls. RESULTS: A total of 83 patients aged 4-52 years were studied; 68.7% had PKU and 31.3% had MHPA. 68 healthy controls of similar sex and age were also evaluated Metabolic control was adequate in 71.9% of PKU patients. Fasting glucose levels (mean 80.77 ± 8.06 mg/dL) were high in just one patient, but fasting insulin levels, with a mean of 12.74 ± 8.4 mIU/L, were altered in 15 PKU patients (26.3%) and markedly higher than in patients with MPHA (p = 0.035). Fasting insulin levels and Homeostasis Model Assessment Insulin Resistance (HOMA-IR) were significantly higher than in healthy controls and correlated with body mass index, waist circumference, age, and also showed statistically significant differences according to diagnosis and Phe tolerance (p < 0.05). Patients under BH4 therapy had lower insulin levels and HOMA-IR. A higher mean carbohydrate intake from AA mixtures was observed in classic PKU patients. The caloric intake in the form of carbohydrates was also higher in PKU than MHPA patients (p = 0.038) and it was correlated with basal insulin (rho = 0.468, p = 0.006), HOMA-IR (rho = 0.423, p = 0.02), BMI (rho 0.533, p = 0.002), and waist circumference (rho 0.584, p = 0.0007). CONCLUSIONS: This study shows that PKU patients are at risk of carbohydrate intolerance and insulin resistance, more evident in adults and overweight patients, probably related to their higher caloric intake in form carbohydrate content. A higher dependency of AA mixtures was demonstrated in PKU patients.

Prioritization of Variants Detected by Next Generation Sequencing According to the Mutation Tolerance and Mutational Architecture of the Corresponding Genes.

The biggest challenge geneticists face when applying next-generation sequencing technology to the diagnosis of rare diseases is determining which rare variants, from the dozens or hundreds detected, are potentially implicated in the patient's phenotype. Thus, variant prioritization is an essential step in the process of rare disease diagnosis. In addition to conducting the usual in-silico analyses to predict variant pathogenicity (based on nucleotide/amino-acid conservation and the differences between the physicochemical features of the amino-acid change), three important concepts should be borne in mind. The first is the "mutation tolerance" of the genes in which variants are located. This describes the susceptibility of a given gene to any functional mutation and depends on the strength of purifying selection acting against it. The second is the "mutational architecture" of each gene. This describes the type and location of mutations previously identified in the gene, and their association with different phenotypes or degrees of severity. The third is the mode of inheritance (inherited vs. de novo) of the variants detected. Here, we discuss the importance of each of these concepts for variant prioritization in the diagnosis of rare diseases. Using real data, we show how genes, rather than variants, can be prioritized by calculating a gene-specific mutation tolerance score. We also illustrate the influence of mutational architecture on variant prioritization using five paradigmatic examples. Finally, we discuss the importance of familial variant analysis as final step in variant prioritization.

A novel missense mutation in GRIN2A causes a nonepileptic neurodevelopmental disorder.

BACKGROUND: Mutations in the GRIN2A gene, which encodes the GluN2A (glutamate [NMDA] receptor subunit epsilon-1) subunit of the N-methyl-d-aspartate receptor, have been identified in patients with epilepsy-aphasia spectrum disorders, idiopathic focal epilepsies with centrotemporal spikes, and epileptic encephalopathies with severe developmental delay. However, thus far, mutations in this gene have not been associated with a nonepileptic neurodevelopmental disorder with dystonia. OBJECTIVES: The objective of this study was to identify the disease-causing gene in 2 siblings with neurodevelopmental and movement disorders with no epileptiform abnormalities. METHODS: The study method was targeted next-generation sequencing panel for neuropediatric disorders and subsequent electrophysiological studies. RESULTS: The 2 siblings carry a novel missense mutation in the GRIN2A gene (p.Ala643Asp) that was not detected in genomic DNA isolated from blood cells of their parents, suggesting that the mutation is the consequence of germinal mosaicism in 1 progenitor. In functional studies, the GluN2A-A643D mutation increased the potency of the agonists L-glutamate and glycine and decreased the potency of endogenous negative modulators, including protons, magnesium and zinc but reduced agonist-evoked peak current response in mammalian cells, suggesting that this mutation has a mixed effect on N-methyl-d-aspartate receptor function. CONCLUSION: De novo GRIN2A mutations can give rise to a neurodevelopmental and movement disorder without epilepsy. © 2018 International Parkinson and Movement Disorder Society.

Similarities between acylcarnitine profiles in large for gestational age newborns and obesity.

Large for gestational age (LGA) newborns have an increased risk of obesity, insulin resistance, and metabolic syndrome. Acylcarnitine profiles in obese children and adults are characterized by increased levels of C3, C5, and certain medium-chain (C12) and long-chain (C14:1 and C16) acylcarnitines. C2 is also increased in insulin-resistant states. In this 1-year observational study of 2514 newborns (246 LGA newborns, 250 small for gestational age (GA) newborns, and 2018 appropriate for GA newborns), we analyzed and compared postnatal acylcarnitine profiles in LGA newborns with profiles described for obese individuals. Acylcarnitine analysis was performed by tandem mass spectrometry on dried-blood spots collected on day 3 of life. LGA newborns had higher levels of total short-chain acylcarnitines (p < 0.001), C2 (p < 0.01) and C3 (p < 0.001) acylcarnitines, and all C12, C14, and C16 acylcarnitines except C12:1. They also had a higher tendency towards carnitine insufficiency (p < 0.05) and carnitine deficiency (p < 0.001). No significant differences were observed between LGA newborns born to mothers with or without a history of gestational diabetes. This novel study describes a postnatal acylcarnitine profile in LGA with higher levels of C2, C3, total acylcarnitines, and total short-chain acylcarnitines that is characteristic of childhood and adult obesity and linked to an unhealthy metabolic phenotype.

Arterial stiffness assessment in patients with phenylketonuria.

In patients with phenylketonuria (PKU) compliant to diet greater tendency to overweight and higher inflammatory biomarkers levels than controls were reported. Although this could lead to atherogenesis, the elastic properties of large arteries in PKU patients have never been assessed. The aim of this study was to assess arterial stiffness measured by applanation tonometry in PKU patients compared to healthy controls.We carried out a cross-sectional study in 41 PKU patients (range age: 6-50 years old) and 41 age- and gender-matched healthy controls. Evaluated data included pharmacological treatment with sapropterin, clinical, and biochemical parameters. Aortic stiffness was assessed noninvasively by applanation tonometry measuring central blood pressure, aortic augmentation index (Aix@HR75), augmentation pressure (AP), and pulse wave velocity (PWV).We found higher PWV in classic PKU patients (6.60 m/second vs 5.26 m/second; P: .044). Percentage of PKU patients with PWV above 90 percentile was higher than controls (14.63% vs 2.32%; P: .048). A positive relationship was observed between the annual Phe median and PWV (r: 0.496; P: .012). PKU subjects with lower Phe tolerance showed more body weight (67.6 kg vs 56.8 kg; P: .012) and more PWV than those with higher Phe tolerance (6.55 m/second vs 5.42 m/second; P: .044).Our data show increased aortic stiffness in PKU patients, measured by applanation tonometry, when compared to healthy controls. Higher Phe levels are associated with a bigger PWV increase, which is not present in those subjects compliant to diet or under sapropterin treatment. These results could have marked effects in both research and clinical daily practice for a proper evaluation of cardiovascular risk in PKU subjects.

Lipid profile status and other related factors in patients with Hyperphenylalaninaemia.

BACKGROUND: The mainstay of treating patients with phenylketonuria (PKU) is based on a Phe-restricted diet, restrictive in natural protein combined with Phe-free L-amino acid supplements and low protein foods. This PKU diet seems to reduce atherogenesis and confer protection against cardiovascular diseases but the results from the few published studies have been inconclusive. The aim of our study was to evaluate the relationship between the lipid profile and several treatment-related risk factors in patients with hyperphenylalaninaemia (HPA) in order to optimize their monitoring. METHODS: We conducted a cross-sectional multicentre study. A total of 141 patients with HPA were classified according to age, phenotype, type of treatment and dietary adherence. Annual median blood phenylalanine (Phe) levels, Phe tolerance, anthropometric measurements, blood pressure (BP) and biochemical parameters [(triglycerides, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), apolipoprotein A (ApoA), apolipoprotein B (ApoB), vitamin B12, total homocysteine (tHcy), Methionine (Met), high sensitivity C-Reactive Protein (hsCRP)] were collected for each patient. RESULTS: Plasma TC levels were lower in patients with PKU than in the mild-HPA group (150 ± 31 vs. 164 ± 22 mg/dL), and there was a weak inverse correlation between plasma TC and Phe levels. HDL-C, LDL-C, ApoA and ApoB levels were lower in the PKU group than in mild-HPA. Patients with PKU had higher systolic BP than the mild-HPA group and there was found a quadratic correlation between median Phe levels and systolic BP (p = 6.42e(-5)) and a linear correlation between median Phe levels and diastolic BP (p = 5.65e(-4)). In overweight or obese PKU patients (24.11 %), biochemical parameters such as TC, triglycerides, LDL-C, tHcy, hsCRP and BP were higher. By contrast, HDL-C was lower in these patients. CONCLUSION: Our data show a direct correlation between lipid profile parameters and good adherence to the diet in PKU patients. However, lipid profile in overweight or obese patients displayed an atherogenic profile, in addition to higher hsCRP concentrations and BP. Our study contributes to a better understanding of the relationship between phenotype and treatment in patients with HPA, which could be useful in improving follow-up strategies and clinical outcome. TRIAL REGISTRATION: Research Ethics Committee of Santiago-Lugo 2015/393. Registered 22 September 2015, retrospectively registered.

Molecular-genetic characterization and rescue of a TSFM mutation causing childhood-onset ataxia and nonobstructive cardiomyopathy.

Oxidative phosphorylation dysfunction has been found in many different disorders. This biochemical pathway depends on mitochondrial protein synthesis. Thus, mutations in components of the mitochondrial translation system can be responsible for some of these pathologies. We identified a new homozygous missense mutation in the mitochondrial translation elongation factor Ts gene in a patient suffering from slowly progressive childhood ataxia and hypertrophic cardiomyopathy. Using cell, biochemical and molecular-genetic protocols, we confirm it as the etiologic factor of this phenotype. Moreover, as an important functional confirmation, we rescued the normal molecular phenotype by expression of the wild-type TSFM cDNA in patient's fibroblasts. Different TSFM mutations can produce the same or very different clinical phenotypes, going from abortions to moderately severe presentations. On the other hand, the same TSFM mutation can also produce same or different phenotypes within the same range of presentations, therefore suggesting the involvement of unknown factors.

Micronutrient in hyperphenylalaninemia.

The data presented here are the biochemical parameters of 156 patients with hyperphenylalaninemia. PKU patients, who, in order to maintain optimal serum Phe concentrations, receive dietary treatment consisting of a diet low in natural protein supplemented with special low protein foods and a Phe-free amino acid mixture, vitamins and minerals. The obtained data reflects a high percentage of patients with prealbumin and selenium deficiencies, as well as an increased level of folic acid. This data article is related to the research article entitled, "Vitamin and mineral status in patients with hyperphenylalaninemia", by Crujeiras et al. [1].

Vitamin and mineral status in patients with hyperphenylalaninemia.

Natural sources of protein and some vitamins and minerals are limited in phenylketonuria (PKU) treated patients, who should receive optimal supplementation although this is not yet fully established. We conducted a cross-sectional observational multicenter study including 156 patients with hyperphenylalaninemia. Patients were stratified by age, phenotype, disease detection and type of treatment. Annual median blood phenylalanine (Phe) levels, Phe tolerance, anthropometric measurements, and biochemical parameters (total protein, prealbumin, electrolytes, selenium, zinc, B12, folic acid, ferritin, 25-OH vitamin D) were collected in all patients. 81.4% of patients had biochemical markers out of recommended range but no clinical symptoms. Total protein, calcium, phosphorus, B12, ferritin, and zinc levels were normal in most patients. Prealbumin was reduced in 34.6% of patients (74% with PKU phenotype and 94% below 18 years old), showing almost all (96.3%) an adequate adherence to diet. Selenium was diminished in 25% of patients (95% with PKU phenotype) and also 25-OHD in 14%. Surprisingly, folic acid levels were increased in 39% of patients, 66% with classic PKU. Phosphorus and B12 levels were found diminished in patients with low adherence to diet. Patients under BH4 therapy only showed significant lower levels of B12. This study shows a high percentage of prealbumin and selenium deficiencies as well as an increased level of folic acid in PKU treated patients, which should lead us to assess an adjustment for standards supplements formulated milks.