Whole-Genome Sequencing Can Identify Clinically Relevant Variants from a Single Sub-Punch of a Dried Blood Spot Specimen.

The collection of dried blood spots (DBS) facilitates newborn screening for a variety of rare, but very serious conditions in healthcare systems around the world. Sub-punches of varying sizes (1.5-6 mm) can be taken from DBS specimens to use as inputs for a range of biochemical assays. Advances in DNA sequencing workflows allow whole-genome sequencing (WGS) libraries to be generated directly from inputs such as peripheral blood, saliva, and DBS. We compared WGS metrics obtained from libraries generated directly from DBS to those generated from DNA extracted from peripheral blood, the standard input for this type of assay. We explored the flexibility of DBS as an input for WGS by altering the punch number and size as inputs to the assay. We showed that WGS libraries can be successfully generated from a variety of DBS inputs, including a single 3 mm or 6 mm diameter punch, with equivalent data quality observed across a number of key metrics of importance in the detection of gene variants. We observed no difference in the performance of DBS and peripheral-blood-extracted DNA in the detection of likely pathogenic gene variants in samples taken from individuals with cystic fibrosis or phenylketonuria. WGS can be performed directly from DBS and is a powerful method for the rapid discovery of clinically relevant, disease-causing gene variants.

Current State and Innovations in Newborn Screening: Continuing to Do Good and Avoid Harm.

In 1963, Robert Guthrie's pioneering work developing a bacterial inhibition assay to measure phenylalanine in dried blood spots, provided the means for whole-population screening to detect phenylketonuria in the USA. In the following decades, NBS became firmly established as a part of public health in developed countries. Technological advances allowed for the addition of new disorders into routine programmes and thereby resulted in a paradigm shift. Today, technological advances in immunological methods, tandem mass spectrometry, PCR techniques, DNA sequencing for mutational variant analysis, ultra-high performance liquid chromatography (UPLC), iso-electric focusing, and digital microfluidics are employed in the NBS laboratory to detect more than 60 disorders. In this review, we will provide the current state of methodological advances that have been introduced into NBS. Particularly, 'second-tier' methods have significantly improved both the specificity and sensitivity of testing. We will also present how proteomic and metabolomic techniques can potentially improve screening strategies to reduce the number of false-positive results and improve the prediction of pathogenicity. Additionally, we discuss the application of complex, multiparameter statistical procedures that use large datasets and statistical algorithms to improve the predictive outcomes of tests. Future developments, utilizing genomic techniques, are also likely to play an increasingly important role, possibly combined with artificial intelligence (AI)-driven software. We will consider the balance required to harness the potential of these new advances whilst maintaining the benefits and reducing the risks for harm associated with all screening.

Cystic fibrosis newborn screening: the importance of bloodspot sample quality.

OBJECTIVE: Wales has an immunoreactive trypsin (IRT)-DNA cystic fibrosis (CF) newborn screening (NBS) programme. Most CF NBS false negative cases are due to an IRT concentration below the screening threshold. The accuracy of IRT results is dependent on the quality of the dried bloodspot (DBS) sample. The aim of this study was to determine the cause of false negative cases in CF NBS and their relationship to DBS quality. DESIGN: Longitudinal birth cohort. SETTING: Wales 1996-2016. PATIENTS: Children with CF. INTERVENTIONS: Identification of all CF patients with triangulation of multiple data sources to detect false negative cases. MAIN OUTCOME MEASURES: False negative cases. RESULTS: Over 20 years, 673 952 infants were screened and 239 were diagnosed with CF (incidence 1:2819). The sensitivity of the programme was 0.958, and positive predictive value was 0.476. Eighteen potential false negatives were identified, of whom eight were excluded: four screened outside Wales, two had complex comorbidities, no identified cystic fibrosis transmembrane conductance regulator (CFTR) variants on extended analysis and thus not considered to have CF and two were diagnosed after their 16th birthday. Of the 10 false negatives, 9 had a low DBS IRT and at least one common CFTR variant and thus should have received a sweat test under the programme. DBS cards were available for inspection for five of the nine false negative cases-all were classified as small/insufficient or poor quality. CONCLUSIONS: The majority of false negatives had a low bloodspot IRT, and this was associated with poor quality DBS. The optimal means to improve the sensitivity of our CF NBS programme would be to improve DBS sample quality.

Newborn screening for homocystinurias: Recent recommendations versus current practice.

PURPOSE: To assess how the current practice of newborn screening (NBS) for homocystinurias compares with published recommendations. METHODS: Twenty-two of 32 NBS programmes from 18 countries screened for at least one form of homocystinuria. Centres provided pseudonymised NBS data from patients with cystathionine beta-synthase deficiency (CBSD, n = 19), methionine adenosyltransferase I/III deficiency (MATI/IIID, n = 28), combined remethylation disorder (cRMD, n = 56) and isolated remethylation disorder (iRMD), including methylenetetrahydrofolate reductase deficiency (MTHFRD) (n = 8). Markers and decision limits were converted to multiples of the median (MoM) to allow comparison between centres. RESULTS: NBS programmes, algorithms and decision limits varied considerably. Only nine centres used the recommended second-tier marker total homocysteine (tHcy). The median decision limits of all centres were ≥ 2.35 for high and ≤ 0.44 MoM for low methionine, ≥ 1.95 for high and ≤ 0.47 MoM for low methionine/phenylalanine, ≥ 2.54 for high propionylcarnitine and ≥ 2.78 MoM for propionylcarnitine/acetylcarnitine. These decision limits alone had a 100%, 100%, 86% and 84% sensitivity for the detection of CBSD, MATI/IIID, iRMD and cRMD, respectively, but failed to detect six individuals with cRMD. To enhance sensitivity and decrease second-tier testing costs, we further adapted these decision limits using the data of 15 000 healthy newborns. CONCLUSIONS: Due to the favorable outcome of early treated patients, NBS for homocystinurias is recommended. To improve NBS, decision limits should be revised considering the population median. Relevant markers should be combined; use of the postanalytical tools offered by the CLIR project (Collaborative Laboratory Integrated Reports, which considers, for example, birth weight and gestational age) is recommended. tHcy and methylmalonic acid should be implemented as second-tier markers.

Identifying Non-Duchenne Muscular Dystrophy-Positive and False Negative Results in Prior Duchenne Muscular Dystrophy Newborn Screening Programs: A Review.

IMPORTANCE: Duchenne muscular dystrophy (DMD) is a candidate for the recommended universal screening panel based on evidence that early corticosteroid treatment improves outcomes and on new genetic therapies that require early diagnosis for effectiveness. Elevated creatine kinase levels in the neonatal period are the initial screening marker in DMD newborn screening programs but is found in inherited muscle disorders other than DMD. Data are needed to inform protocols for future screening and follow-up testing and care in these patients. OBJECTIVES: To review non-DMD muscle disorders identified by prior DMD screening programs and to investigate whether these programs failed to identify patients later diagnosed as having DMD (false-negative findings). EVIDENCE REVIEW: Since 1975, 10 DMD newborn screening programs have provided opportunities to study screening protocols, outcomes, and parental responses. These programs used elevated creatine kinase levels in dried blood spots for the initial screening, with the diagnosis of DMD based on findings of clinical follow-up, muscle biopsy, or direct mutational testing of the DMD gene. Literature regarding these prior programs was reviewed in PubMed, and the programs were discussed directly with the directors when possible to identify diagnoses of non-DMD disorders and false negative results from 1975 to July 12, 2015. Data were collected from screening programs, which were active between 1975 and December 2011. Data were analyzed from March 26, 2015, to August 24, 2015. FINDINGS: The 10 screening programs screened more than 1.8 million newborns between 1975 and 2011, and 344 were diagnosed with DMD. Of those screened, the majority were boys. Across all programs, 80 patients had positive results for non-DMD disorders, including Becker muscular dystrophy and forms of limb-girdle and congenital muscular dystrophies, and 21 patients had false-negative findings for DMD. CONCLUSIONS AND RELEVANCE: Screening for DMD will result in identification of other muscle diseases. Future screening protocols should include infants of both sexes and include follow-up testing algorithms to evaluate patients who do not have DMD gene mutations but may have another muscle disorder associated with elevated neonatal creatine kinase levels. These programs will need to be aware that false-negative results are a possibility.

Newborn bloodspot screening for Duchenne muscular dystrophy: 21 years experience in Wales (UK).

Duchenne muscular dystrophy (DMD), a progressive X-linked neuromuscular disorder, has an estimated worldwide incidence of 1:3500 male births. Currently, there are no curative treatments and the mean age of diagnosis is 5 years. In addition, subsequent pregnancies frequently occur before a diagnosis is made in an index case. An 'opt in' screening programme was introduced in Wales in 1990 with the aim to: reduce the diagnostic delay, permit reproductive choice and allow planning of the care of the affected boy. Newborn bloodspots were collected routinely as part of the Wales newborn screening programme. Specific consent was obtained for this test separately from the other tests. During the 21-year period, 369,780 bloodspot cards were received from male infants, of these 343,170 (92.8%) were screened using a bloodspot creatine kinase (CK) assay following parental consent. A total of 145 cases had a raised CK activity (≥250 U/l) and at follow-up, at 6-8 weeks of age, 79 cases had a normal serum CK (false-positive rate 0.023%) and 66 cases had an elevated serum CK. DMD was confirmed in 56 cases by genotyping/muscle biopsy studies, Becker muscular dystrophy in 5 cases and other rarer forms of muscular dystrophy in 5 cases. This long-term study has so far identified 13 false-negative cases. The incidence of DMD in Wales of 1:5136 during this period is lower than that of 1:4046 before commencement of screening in Wales. Screening has reduced the diagnostic delay enabling reproductive choice for parents of affected boys and earlier administration of current therapies.

A vitamin B-12 supplement of 500 µg/d for eight weeks does not normalize urinary methylmalonic acid or other biomarkers of vitamin B-12 status in elderly people with moderately poor vitamin B-12 status.

Plasma vitamin B-12 is the most commonly used biomarker of vitamin B-12 status, but the predictive value for low vitamin B-12 status is poor. The urinary methylmalonic acid (uMMA) concentration has potential as a functional biomarker of vitamin B-12 status, but the response to supplemental vitamin B-12 is uncertain. A study was conducted to investigate the responsiveness of uMMA to supplemental vitamin B-12 in comparison with other biomarkers of vitamin B-12 status [plasma vitamin B-12, serum holotranscobalamin (holoTC), plasma MMA] in elderly people with moderately poor vitamin B-12 status. A double-blind, placebo-controlled, randomized 8-wk intervention study was carried out using vitamin B-12 supplements (500 µg/d, 100 µg/d, and 10 µg/d cyanocobalamin) in 100 elderly people with a combined plasma vitamin B-12 <250 pmol/L and uMMA ratio (µmol MMA/mmol creatinine) >1.5. All biomarkers had a dose response to supplemental vitamin B-12. Improvements in plasma vitamin B-12 and serum holoTC were achieved at cobalamin supplements of 10 µg/d, but even 500 µg/d for 8 wk did not normalize plasma vitamin B-12 in 8% and serum holoTC in 12% of people. The response in uMMA was comparable with plasma MMA; 15-25% of people still showed evidence of metabolic deficiency after 500 µg/d cobalamin for 8 wk. There was a differential response in urinary and plasma MMA according to smoking behavior; the response was enhanced in ex-smokers compared with never-smokers. uMMA offers an alternative marker of metabolic vitamin-B12 status, obviating the need for blood sampling.

Differential effects of low and high dose folic acid on endothelial dysfunction in a murine model of mild hyperhomocysteinaemia.

The exact mechanism(s) by which hyperhomocysteinaemia promotes vascular disease remains unclear. Moreover, recent evidence suggests that the beneficial effect of folic acid on endothelial function is independent of homocysteine-lowering. In the present study the effect of a low (400 microg/70 kg/day) and high (5 mg/70 kg/day) dose folic acid supplement on endothelium-dependent relaxation in the isolated perfused mesenteric bed of heterozygous cystathionine beta-synthase deficient mice was investigated. Elevated total plasma homocysteine and impaired relaxation responses to methacholine were observed in heterozygous mice. In the presence of N(G)-nitro-L-arginine methyl ester relaxation responses in wild-type tissues were reduced, but in heterozygous tissues were abolished. Clotrimazole and 18alpha-glycyrrhetinic acid, both inhibitors of non-nitric oxide/non-prostanoid-induced endothelium-dependent relaxation, reduced responses to methacholine in wild-type but not heterozygous tissues. The combination of N(G)-nitro-L-arginine methyl ester and either clotrimazole or 18alpha-glycyrrhetinic acid completely inhibited relaxation responses in wild-type tissues. Both low and high dose folic acid increased plasma folate, reduced total plasma homocysteine and reversed endothelial dysfunction in heterozygous mice. A greater increase in plasma folate in the high dose group was accompanied by a more significant effect on endothelial function. In the presence of N(G)-nitro-L-arginine methyl ester, a significant residual relaxation response was evident in tissues from low and high dose folic acid treated heterozygous mice. These data suggest that the impaired mesenteric relaxation in heterozygous mice is largely due to loss of the non-nitric oxide/non-prostanoid component. While low dose folic acid may restore this response in a homocysteine-dependent manner, the higher dose has an additional effect on nitric oxide-mediated relaxation that would appear to be independent of homocysteine lowering.

Homocysteine and endothelial function in human studies.

The endothelium plays a key role in the pathophysiology of vascular disease. Impaired flow-mediated dilatation (FMD) is a measure of endothelial dysfunction resulting from reduced bioavailability of nitric oxide (NO). Patients with homocystinuria manifest with impaired FMD, but in mild hyperhomocysteinemia, the evidence is conflicting. Oral loading with methionine or homocysteine impairs FMD, but it remains unproven that this effect is mediated directly by homocysteine. In addition, there is no clear consensus as to a mechanisms by which homocysteine would induce endothelial dysfunction. Folate administration lowers plasma homocysteine and enhances FMD. However, the effect of folate only appears to occur at high doses and with a time course that would indicate that it is acting by a mechanism independent of homocysteine lowering. It is possible that folate, in pharmacological doses, may enhance the NO activity by influencing NO-tetrahydrobiopterin interactions. These studies provide some insights and raise intriguing questions concerning the relationship between homocysteine, folate, and endothelial function. However, changes in FMD may not translate into vascular endpoints, and the outcomes of clinical intervention trials with different doses of folic acid are awaited with interest.

Short-term high-dose folic acid does not alter markers of endothelial cell damage in patients with coronary heart disease.

BACKGROUND AND OBJECTIVE: Endothelial dysfunction is an early, pre-clinical manifestation of coronary heart disease and is associated with increased plasma levels of von Willebrand factor (vWF), soluble E-selectin, and thrombomodulin, markers of endothelial cell damage/activation and reduced nitric oxide bioavailability. Homocysteine is associated with an increased risk of cardiovascular disease and mortality. High-dose folic acid treatment lowers plasma homocysteine by 25% and improves nitric oxide bioavailability; however, the effects on other indices of endothelial cell activation/damage has not been examined in patients with coronary heart disease and normal renal function. DESIGN AND METHODS: In a randomised, double-blind, cross-over study in 50 patients with coronary heart disease and normal serum creatinine, folic acid (5 mg/daily) was administered for 6 weeks and blood was analysed for von Willebrand factor, soluble E-selectin, and thrombomodulin. Endothelial nitric oxide bioavailability was assessed by flow-mediated dilatation. RESULTS: Plasma folate levels increased (9.1+/-3.4 vs. 310+/-235 microg/l; p<0.001) and nitric oxide bioavailability improved (47+/-35 vs. 110+/-43 microm; p<0.001) following active treatment. However, markers of endothelial cell injury were not significantly influenced (von Willebrand factor 118+/-33 vs. 119+/-34%; E-selectin 52+/-17 vs. 51+/-16 microg/l; thrombomodulin 3.94+/-1.81 vs. 3.94+/-1.51 microg/l; p=NS comparing post-placebo with post-folate). No correlation was observed between improvement in flow-mediated dilatation and change in endothelial marker proteins. INTERPRETATION AND CONCLUSION: These data suggest that endothelial markers are not useful surrogates of endothelial nitric oxide bioavailability in coronary heart disease and may be a less sensitive marker of endothelial function than nitric oxide.

The molecular basis of cystathionine beta-synthase (CBS) deficiency in UK and US patients with homocystinuria.

The molecular basis of cystathionine beta-synthase (CBS) deficiency has been studied in 536 patient alleles with 130 different mutations described. To date, no study has reported on the incidence of any of the reported mutations in patients from the UK and the US. We developed a new antisense oligonucleotide (ASO) PCR/hybridization method to screen for 12 of the most frequent CBS mutations in 14 unrelated patients from the UK and 38 unrelated patients from the US, a total of 104 independent alleles. We determined 16/28 (57%) and 28/76 (37%) of the affected alleles in the UK and US patients, respectively. Four different mutations were identified in the UK patients (c.374G>A, R125Q; c.430G>A, E144K; c.833T>C, I278T; c.919G>A, G307S) and 8 mutations identified in the patients from the US (c.341C>T, A114V; c.374G>A, R125Q; c.785C>T, T262M; c.797G>A, R266K; c.833T>C, I278T; c.919G>A, G307S; g.13217A>C (del ex 12); c.1330G>A, D444N). The I278T was the predominant mutation in both populations, present in 8 (29%) of 28 independent alleles from the UK and in 14 (18%) of 76 independent alleles from the US. The incidence of the G307S mutation was 21% in the UK patients and 8% in the US patients. The spectrum of mutations observed in the patients from the UK and US is closer to that which is observed in Northern Europe and bears less resemblance to that observed in Ireland.

An indirect response model of homocysteine suppression by betaine: optimising the dosage regimen of betaine in homocystinuria.

AIMS: To investigate the pharmacokinetics (PK) and pharmacodynamics (PD) of betaine in the treatment of classical homocystinuria due to cystathionine beta-synthase (CbetaS) deficiency with a view to optimizing the dosage regimen. METHODS: Betaine was given as a single oral dose of 100 mg kg(-1) to six patients (age range 6-17 years) who normally received betaine but whose treatment had been suspended for 1 week prior to the study. Plasma betaine and total homocysteine concentrations were measured by high performance liquid chromatography (h.p.l.c.) at frequent intervals over 24 h. The best-fit PK model was determined using the PK-PD program Win-Nonlin and the concentration-time-effect data analysed by an indirect PD model. Using the PK and PD parameters, simulations were carried out with the aim of optimizing betaine dosage. RESULTS: Betaine PK was described by both mono- and bi-exponential disposition functions with first order absorption and a lag time. The correlation coefficient between betaine oral clearance and body weight was 0.6. Mean betaine clearance was higher in males than in females (P=0.03). PK-PD simulation indicated minimal benefit from exceeding a twice-daily dosing schedule and a 150 mg kg(-1) day(-1) dosage for betaine. CONCLUSIONS: PK-PD modelling allows recommendations for optimal dosage of betaine in the treatment of homocystinuria, that have the potential for improved patient compliance and both therapeutic and pharmacoeconomic benefit.

Folic acid improves endothelial function in coronary artery disease via mechanisms largely independent of homocysteine lowering.

BACKGROUND: Homocysteine is a risk factor for coronary artery disease (CAD), although a causal relation remains to be proven. The importance of determining direct causality rests in the fact that plasma homocysteine can be safely and inexpensively reduced by 25% with folic acid. This reduction is maximally achieved by doses of 0.4 mg/d. High-dose folic acid (5 mg/d) improves endothelial function in CAD, although the mechanism is controversial. It has been proposed that improvement occurs through reduction in total (tHcy) or free (non-protein bound) homocysteine (fHcy). We investigated the effects of folic acid on endothelial function before a change in homocysteine in patients with CAD. METHODS AND RESULTS: A randomized, placebo-controlled study of folic acid (5 mg/d) for 6 weeks was undertaken in 33 patients. Endothelial function, assessed by flow-mediated dilatation (FMD), was measured before, at 2 and 4 hours after the first dose of folic acid, and after 6 weeks of treatment. Plasma folate increased markedly by 1 hour (200 compared with 25.8 nmol/L; P<0.001). FMD improved at 2 hours (83 compared with 47 microm; P<0.001) and was largely complete by 4 hours (101 compared with 51 microm; P<0.001). tHcy did not significantly differ acutely (4-hour tHcy, 9.56 compared with 9.79 micromol/L; P=NS). fHcy did not differ at 3 hours but was slightly reduced at 4 hours (1.55 compared with 1.78 micromol/L; P=0.02). FMD improvement did not correlate with reductions in either fHcy or tHcy at any time. CONCLUSIONS: These data suggest that folic acid improves endothelial function in CAD acutely by a mechanism largely independent of homocysteine.