Newborn screening (NBS) for congenital hypothyroidism (CH) started in the 1970s, with the introduction of radioimmuno assays (RIA) for the measurement of thyroxine (T4), and thyroid stimulating ...hormone (TSH). With the development of sensitive enzyme immune assays (EIA, FIA, FEIA), RIAs were replaced in the newborn screening laboratories. With the increasing number of analytes and centralization of NBS, there is a growing demand of total automation. In the course of method validation, two fully automated platforms for the determination of TSH in dried blood samples (DBS) were compared. The GSP from PerkinElmer (PE), and the NS2400 from Labsystems (LDx), together with the recommended test kits from both manufacturers. Both systems showed good performance, with recoveries, of 103.0% (LDx) and 98.5% (PE), and CVs for intra and interassay variations at various concentrations, between 4.3 and 15.7. Both assays had a good correlation (r2 = 0.8814). With LDx/NS2400 platform, TSH values were in the mean 2.09 mU/L higher; however, the difference of both results from the mean was within ±2 SD, up to 30 mU/L, and only for values above 50 mU/L did the difference become bigger. However, this has no influence on the clinical interpretation. No false negative results were observed with either of the two platforms. TSH results obtained with the LDx/NS2400 were slightly higher than those obtained with the PE/GSP; however, the recall rate was lower: 0.059% compared to 0.063%. This can be explained by the much narrower distribution of TSH values. In conclusion, both platforms are equally suitable for medium and large NBS laboratories. However, due to the more open structure the LDx/NS2400 platform has a lot of advantages compared to the totally closed PE/GSP platform.
CRISPR-Cas-based genome editing holds great promise for targeting genetic disorders, including inborn errors of hepatocyte metabolism. Precise correction of disease-causing mutations in adult tissues ...in vivo, however, is challenging. It requires repair of Cas9-induced double-stranded DNA (dsDNA) breaks by homology-directed mechanisms, which are highly inefficient in nondividing cells. Here we corrected the disease phenotype of adult phenylalanine hydroxylase (Pah)
mice, a model for the human autosomal recessive liver disease phenylketonuria (PKU)
, using recently developed CRISPR-Cas-associated base editors
. These systems enable conversion of C∙G to T∙A base pairs and vice versa, independent of dsDNA break formation and homology-directed repair (HDR). We engineered and validated an intein-split base editor, which allows splitting of the fusion protein into two parts, thereby circumventing the limited cargo capacity of adeno-associated virus (AAV) vectors. Intravenous injection of AAV-base editor systems resulted in Pah
gene correction rates that restored physiological blood phenylalanine (L-Phe) levels below 120 µmol/l 5. We observed mRNA correction rates up to 63%, restoration of phenylalanine hydroxylase (PAH) enzyme activity, and reversion of the light fur phenotype in Pah
mice. Our findings suggest that targeting genetic diseases in vivo using AAV-mediated delivery of base-editing agents is feasible, demonstrating potential for therapeutic application.
Newborn screening (NBS) for congenital hypothyroidism (CH) was introduced in Switzerland in 1977, which allowed for the preclinical, biochemical diagnosis. The aim of this study was to evaluate the ...prevalence of transient CH (tCH) in the canton of Zurich. In this analytical cohort study, all newborns born in the canton of Zurich, between the 1st of January 2000 and the 30st of June 2016, with a TSH value above 15 mU/L (whole blood) were included. There were 115 cases out of 247,918 babies born during the study period. However, 23 cases had to be excluded due to missing data. The definite diagnosis was made after a thyroxine withdrawal at 2 years of age. The total prevalence of confirmed CH and the female to male ratio (f/m) were 1:2695 and 2.17:1; for permanent CH (pCH), 1:3443 and 2.8:1; and for tCH, 1:12,396 and 1:1, respectively. The TSH value was significantly higher in pCH compared to tCH, at 130.3 (62.9-171.9) and 36.4 (26.5-53.3) (median and interquartile range), respectively (
< 0.001). The prevalences found for congenital hypothyroidism and its transient form are comparable to previous studies. TSH concentration at birth was predictive for the further course of the disease. Low birth weight correlated with a tCH, whereas low gestational age did not. The dominance of the female sex in congenital hypothyroidism is supported by a gender ratio of 2.17:1.
The urea cycle disorder argininemia is caused by a defective arginase 1 (ARG1) enzyme resulting from mutations in the ARG1 gene. Patients generally develop hyperargininemia, spastic paraparesis, ...progressive neurological and intellectual impairment, and persistent growth retardation. Interestingly, in contrast to other urea cycle disorders, hyperammonemia is rare. We report here 66 mutations (12 of which are novel), including 30 missense mutations, seven nonsense, 10 splicing, 15 deletions, two duplications, one small insertion, and one translation initiation codon mutation. For the most common mutations (p.Thr134Ile, p.Gly235Arg and p.Arg21*), which cluster geographically in Brazil, China, or Turkey, a structural rationalization of their effect has been included. In order to gain more knowledge on the disease, we have collected clinical and biochemical information of 112 patients, including the patients’ genetic background and ethnic origin. We have listed as well the missense variants with unknown relevance. For all missense variants (of both known and unknown relevance), the conservation, severity prediction, and ExAc scores have been included. Lastly, we review ARG1 regulation, animal models, diagnostic strategies, newborn screening, prenatal testing, and treatment options.
Patients with the urea cycle disorder argininemia generally develop hyperargininemia, spastic paraparesis, progressive neurological and intellectual impairment and persistent growth retardation. We report here 66 mutations (12 of which are novel), including 30 missense mutations, 7 nonsense, 10 splicing, 15 deletions, two duplications, one small insertion and one translation initiation codon mutation. As well, clinical and biochemical information of the 112 patients, including the patients' genetic background and ethnic origin. Lastly, we list missense variants with unknown relevance.
Increased propionylcarnitine levels in newborn screening are indicative for a group of potentially severe disorders including propionic acidemia (PA), methylmalonic acidemias and combined ...remethylation disorders (MMACBL). This alteration is relatively non-specific, resulting in the necessity of confirmation and differential diagnosis in subsequent tests. Thus, we aimed to develop a multiplex approach for concurrent determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid from the same dried blood spot (DBS) as in primary screening (second-tier test). We also set out to validate the method using newborn and follow-up samples of patients with confirmed PA or MMACBL.
The assay was developed using liquid chromatography-tandem mass spectrometry and clinically validated with retrospective analysis of DBS samples from PA or MMACBL patients.
Reliable determination of all three analytes in DBSs was achieved following simple and fast (<20 min) sample preparation without laborious derivatization or any additional pipetting steps. The method clearly distinguished the pathological and normal samples and differentiated between PA and MMACBL in all stored newborn specimens. Methylcitric acid was elevated in all PA samples; 3-hydroxypropionic acid was also high in most cases. Methylmalonic acid was increased in all MMACBL specimens; mostly together with methylcitric acid.
A liquid chromatography-tandem mass spectrometry assay allowing simultaneous determination of the biomarkers 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in DBSs has been developed. The assay can use the same specimen as in primary screening (second-tier test) which may reduce the need for repeated blood sampling. The presented preliminary findings suggest that this method can reliably differentiate patients with PA and MMACBL in newborn screening. The validated assay is being evaluated prospectively in a pilot project for extension of the German newborn screening panel (‟Newborn screening 2020"; Newborn Screening Center, University Hospital Heidelberg).
Key points
Lat4 (Slc43a2) transports branched‐chain amino acids, phenylalanine and methionine, and is expressed in kidney tubule and small intestine epithelial cells.
Using a new knockout model as a ...negative control, it is shown that Lat4 is expressed at the basolateral side of small intestine enterocytes and kidney epithelial cells of the proximal tubule, thick ascending limb and distal convoluted tubule.
In the Xenopus oocyte expression system, Lat4 is shown to function as a uniporter with symmetric intracellular and extracellular apparent affinities for phenylalanine.
Mice lacking Lat4 display a slight intrauterine growth restriction, postnatal malnutrition and early death, presumably as a result of defective amino acid (re)absorption.
These results demonstrate the crucial role that the uniporter Lat4 plays for amino acid transport across cellular barriers and mouse development.
Amino acid (AA) uniporter Lat4 (Slc43a2) mediates facilitated diffusion of branched‐chain AAs, methionine and phenylalanine, although its physiological role and subcellular localization are not known. We report that Slc43a2 knockout mice were born at expected Mendelian frequency but displayed an ∼10% intrauterine growth retardation and low amniotic fluid AAs, suggesting defective transplacental transport. Postnatal growth was strongly reduced, with premature death occurring within 9 days such that further investigations were made within 3 days of birth. Lat4 immunofluorescence showed a strong basolateral signal in the small intestine, kidney proximal tubule and thick ascending limb epithelial cells of wild‐type but not Slc43a2 null littermates and no signal in liver and skeletal muscle. Experiments using Xenopus laevis oocytes demonstrated that Lat4 functioned as a symmetrical low affinity uniporter with a K0.5 of ∼5 mm for both in‐ and efflux. Plasma AA concentration was decreased in Slc43a2 null pups, in particular that of non‐essential AAs alanine, serine, histidine and proline. Together with an increased level of plasma long chain acylcarnitines and a strong alteration of liver gene expression, this indicates malnutrition. Attempts to rescue pups by decreasing the litter size or by nutrients injected i.p. did not succeed. Radioactively labelled leucine but not lysine given per os accumulated in the small intestine of Slc43a2null pups, suggesting the defective transcellular transport of Lat4 substrates. In summary, Lat4 is a symmetrical uniporter for neutral essential AAs localizing at the basolateral side of (re)absorbing epithelia and is necessary for early nutrition and development.
Congenital disorders of glycosylation (CDG) are a growing group of inherited diseases causing manifold symptoms. Routine diagnostic procedures are high performance liquid chromatography (HPLC) or ...isoelectric focusing (IEF) of serum transferrin.
We introduce a modified method to screen for glycosylation abnormalities from dried blood spot (DBS) samples based on isoelectric focusing. In PGM1-CDG, glycosylation analysis and enzyme activity measurement were performed from a single DBS sample. Furthermore, we present the possibility to use capillary blood samples for quantification of transferrin isoforms.
IEF from DBS samples is possible and results are identical to the ones obtained in serum samples. Gel analysis using the ImageJ software allows quantification of IEF results. Storage at −20 °C ensures stable samples for more than six months. Capillary blood samples are equally suitable for glycosylation analysis and show no inferiority to serum samples.
In view of a growing number of treatable CDG subtypes, the proposed methods allow reliable diagnosis and therapy control of CDG while being easily applicable. Capillary blood samples can be taken at home and sent in for follow-up. DBS are widely used in new-born screening programs and have the potential to broaden the knowledge of glycosylation abnormalities in early infancy. By its possible application in the context of alcohol abuse, the proposed method bears the potential for widespread use in a non-metabolic context.
•Serum transferrin is a biomarker for glycosylation disorders and alcohol abuse.•Transferrin glycosylation analysis is possible from dried blood spots.•Storage at −20 °C results in stable samples for more than six months.•Capillary blood samples are suitable for glycosylation analysis by HPLC.
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