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Background: Many newborn screening programs worldwide have introduced screening for diseases using DNA extracted from dried blood spots (DBS). In Germany, DNA-based assays are currently used to screen for severe combined immunodeficiency (SCID), spinal muscular atrophy (SMA), and sickle cell disease (SCD)., Methods: This study analysed the impact of pre-analytic DNA carry-over in sample preparation on the outcome of DNA-based newborn screening for SCID and SMA and compared the efficacy of rapid extraction versus automated protocols. Additionally, the distribution of T cell receptor excision circles (TREC) on DBS cards, commonly used for routine newborn screening, was determined., Results: Contaminations from the punching procedure were detected in the SCID and SMA assays in all experimental setups tested. However, a careful evaluation of a cut-off allowed for a clear separation of true positive polymerase chain reaction (PCR) amplifications. Our rapid in-house extraction protocol produced similar amounts compared to automated commercial systems. Therefore, it can be used for reliable DNA-based screening. Additionally, the amount of extracted DNA significantly differs depending on the location of punching within a DBS., Conclusions: Newborn screening for SMA and SCID can be performed reliably. It is crucial to ensure that affected newborns are not overlooked. Therefore a carefully consideration of potential contaminating factors and the definition of appropriate cut-offs to minimise the risk of false results are of special concern. It is also important to note that the location of punching plays a pivotal role, and therefore an exact quantification of TREC numbers per μl may not be reliable and should therefore be avoided., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bzdok et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Sickle cell disease (SCD) is a group of hemoglobinopathies with a common point mutation causing the production of sickle cell hemoglobin (HbS). In high-throughput newborn screening (NBS) for SCD, a two-step procedure is suitable, in which qPCR first pre-selects relevant samples that are differentiated by a second method., Methods: Three NBS centers using qPCR-based primary screening for SCD performed a laboratory comparison. Methods using tandem MS or HPLC were used for differentiation., Results: In a benchmarking test, 450 dried blood samples were analyzed. Samples containing HbS were detected as reliably by qPCR as by methods established for hemoglobinopathy testing. In a two-step screening approach, the 2 nd -tier-analyses have to distinguish the carrier status from pathological variants. In nine months of regular screening, a total of 353,219 samples were analyzed using two-stage NBS procedures. The 1 st -tier screening by qPCR reduced the number of samples for subsequent differentiation by>99.5%. Cases with carrier status or other variants were identified as inconspicuous while 78 cases with SCD were revealed. The derived incidence of 1:4,773, is in good agreement with previously published incidences., Conclusion: In high-throughput NBS for SCD, qPCR is suitable to focus 2 nd -tier analyses on samples containing HbS, while being unaffected by factors such as prematurity or transfusions. The substantial reduction of samples numbers positively impacts resource conservation, sustainability, and cost-effectiveness. No false negative cases came to attention., Competing Interests: The authors declare that they have no conflict of interest., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Now that targeted therapies for spinal muscular atrophy are available, attempts are being made worldwide to include screening for spinal muscular atrophy in general newborn screening. In Germany, after pilot projects from 2018-2021, it was included in the general newborn screening from October 2021. To ensure a smooth transition, criteria for follow-up were developed together with key stakeholders. At the beginning of the transition to nationwide screening, false positive findings were reported in 3 patients. After optimization of the screening method in the laboratories concerned, all findings have been subsequently confirmed. On average, the first presentation to a neuromuscular center occurred on day 12 of life, and in patients with 2 or 3 SMN2 copies, therapy started on day 26 of life. Compared with the pilot project, there was no significant delay in timing.

Objective: Fast and reliable detection of infection is a key to control the SARS-CoV-2 pandemic. Lateral flow antigen tests (LFATs) are inexpensive, easy to use, but have to be verified, as they are rather unspecific and can produce both, false positive and false negative results. Our objective was to combine the speed of LFAT for SARS-CoV-2 with the reliability of qPCR tests., Methods: A serial dilution of a patient sample positive for SARS-CoV-2 was prepared and added to LFAT wells from two manufacturers. After evaluation, the devices were opened, the strips removed and extracted in a solution. Amplification was performed using point of care PCR systems (cobas® Liat®, ID NOW™) or on a LightCycler after extraction by MagNAPure 96., Results: The nucleic acid amplification systems yielded higher sensitivity to LFAT. Thus, all samples determined positive by LFAT from the serial dilution were also positive in the subsequent amplification reactions. Sensitivity using extracted eluates was 10-100 times higher., Significance: The usage of LFAT is highly recommended for single samples in emergency dental or emergency clinical settings, for smaller cohorts, or even for larger population screening, as it is inexpensive and fast. Positive results can be conveniently verified directly from the test devices using either point of care test equipment or more complex laboratory equipment thus making a major impact on efficient management of infections and isolations., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In 2013 we presented results showing that at the histological level lifelong increased physical activity promotes reinnervation of muscle fibers in aging muscles. Indeed, in muscle biopsies from 70-year old men with a lifelong history of high-level physical activity, we observed a considerable increase in fiber-type groupings (F-TG), almost exclusively of the slow type. Slow-type transformation by denervation-reinnervation in senior sportsmen seems to fluctuate from those with scarce fiber-type transformation and groupings to almost fully transformed muscle, going through a process in which isolated fibers co-expressing fast and slow Myosin Heavy Chains (MHCs) seems to fill the gaps. Taken together, our results suggest that, beyond the direct effects of aging on the muscle fibers, changes occurring in skeletal muscle tissue appear to be largely, although not solely, a result of sparse denervation-reinnervation. The lifelong exercise allows the body to adapt to the consequences of the age-related denervation and to preserve muscle structure and function by saving otherwise lost muscle fibers through recruitment to different, mainly slow, motor units. These beneficial effects of high-level life-long exercise on motoneurons, specifically on the slow type motoneurones that are those with higher daily activity, and on muscle fibers, serve to maintain size, structure and function of muscles, delaying the functional decline and loss of independence that are commonly seen in late aging. Several studies of independent reserchers with independent analyses confirmed and cited our 2013 results. Thus, the results we presented in our paper in 2013 seem to have held up rather well.

Background: Prompt treatment after genetic NBS for SMA substantially improves outcome in infantile SMA. However, deficiency of SMN-protein can cause damage of motor neurons even prior to birth., Objective: To describe the neurological status at the time of NBS and the reversibility of neurological deficits in a cohort of patients with only two copies of the SMN2 gene., Methods: We present motor, respiratory, and bulbar outcomes of 21 SMA patients identified in newborn screening projects in Germany. Inclusion criteria was initiation of SMN targeted medication at less than 6 weeks of age and a minimum age of 9 months at last examination., Results: Twelve patients (57%) developed completely normally, reaching motor milestones in time and having no bulbar or respiratory problems. Three children (14.5%) caught up after initial delay in motor development. Six patients (29%) developed proximal weakness despite early treatment: Three of them (14.5%) achieved the ability to walk with assistance and the other three (14.5%) showed an SMA type 2 phenotype at the age of 16-30 months. One patient (4.8%) had respiratory problems. Three children (14.5%) had mild chewing problems and two individuals (9.5%) needed feeding via gastrotube. Initial CHOP-INTEND values below 30 could be indicative of a less favourable outcome, whereas values above 50 could indicate a good outcome, however in-depth statistic due to the small case number is not predictive., Conclusion: More than 70% of SMA patients with two SMN2 copies can achieve independent ambulation with immediate initiation of therapy. However, caregivers and paediatricians must be informed about the possibility of less favourable outcomes when discussing therapeutic strategies.

Background: Spinal muscular atrophy (SMA) is the most common neurodegenerative disease in childhood. Since motor neuron injury is usually not reversible, early diagnosis and treatment are essential to prevent major disability. Our objective was to assess the impact of genetic newborn screening for SMA on outcome., Methods: We provided clinical data from 43 SMA patients, identified via polymerase chain reaction of the SMN1 gene from dried blood spots between January 2018 and January 2020 in Germany. Follow-up included neurophysiological examinations and standardized physiotherapeutic testing., Results: Detection of SMA with newborn screening was consistent with known incidence in Germany. Birth prevalence was 1:6910; 39.5% had 2 SMN2 copies, 23% had 3 SMN2 copies, 32.5% had 4 copies, and 4.5% had 5 copies of the SMN2 gene. Treatment with SMA-specific medication could be started at the age of 14-39 days in 21 patients. Pre-symptomatically treated patients remained throughout asymptomatic within the observation period. 47% of patients with 2 SMN2 copies showed early, presumably intrauterine onset of disease. These patients reached motor milestones with delay; none of them developed respiratory symptoms. Untreated children with 2 SMN2 copies died. Untreated children with 3 SMN2 copies developed proximal weakness in their first year. In patients with ≥ 4 SMN2 copies, a follow-up strategy of "watchful waiting" was applied despite the fact that one of them was treated from the age of 6 months. Two infant siblings with 4 SMN2 copies were identified with a missed diagnosis of SMA type 3., Conclusion: Identification of newborns with infantile SMA and prompt SMA-specific treatment substantially improves neurodevelopmental outcome, and we recommend implementation in the public newborn screening in countries where therapy is available. Electrophysiology is a relevant parameter to support the urgency of therapy. There has to be a short time interval between a positive screening result and referral to a therapy-ready specialized treatment center.

Objective: The aim is to recommend a fast and cost-effective screening procedure for UK/SA SARS-CoV-2 variants in a routing diagnostic setting., Methods: A rapid procedure using qPCR is described to provide clinicians with information about the two currently most prevalent variants (B1.1.7 and B1.351) that harbour receptor binding domain mutation N501Y. The N501Y specific assay only delivers an amplification signal if the Y501 variant is present., Results: 436 samples initially screened positive for SARS-CoV-2 were randomly selected. Only one of these samples showed a fluorescence signal increase indicative for the Y501 variant. The remaining 435 samples had a melting peak at 54 °C indicating the N501 wildtype., Significance: The screening of a broad population base can still be performed with the established test system. In case of a positive test for SARS-CoV-2 and corresponding clinical and anamnestic indications, a second qPCR for the mutation N501Y can follow and deliver the result to public health authorities and to the treating physician within a few hours., (Copyright © 2021. Published by Elsevier Inc.)

Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disorder, which causes loss of renal proximal tubular function and progressive loss of glomerular function, finally leading to end stage renal failure at school age. In the course of the disease most patients will need kidney transplantation if treatment has not been started before clinical manifestation. With an effective treatment available, a newborn screening assay is highly demanded. Since newborns with cystinosis usually do not show symptoms within the first months of life and no biochemical markers are easily detectable, a DNA-based method seems to be an obvious tool for early diagnosis. Screening was performed using high-throughput nucleic acid extraction followed by 384-well qPCR and melting analysis for the three most frequent variants (57 kb deletion NC_000017.11:g.3600934_3658165del (GRCh38); c.18_21del GACT; c.926dupG) responsible for the defective lysosomal membrane protein cystinosin (CTNS). To increase sensitivity, all heterozygous samples identified in qPCR assay were verified and screened for additional variants by applying next generation sequencing. From January 2018 to July 2019 nearly 292,000 newborns were successfully screened. We identified two newborns with a homozygous 57 kb deletion and a second one with heterozygous 57 kb deletion and a G>C substitution at position c.-512 on the second allele. Cystinosis is an example for diseases caused by a limited number of high prevalence and a high number of low prevalence variants. We have shown that qPCR combined with NGS can be used as a high throughput, cost effective tool in newborn screening for such diseases.

Establishing nucleic acid-based assays for genetic newborn screening (NBS) provides the possibility to screen for genetically encoded diseases like spinal muscular atrophy (SMA), best before the onset of symptoms. Such assays should be easily scalable to 384-well reactions that make the screening of up to 2000 samples per day possible. We developed a test procedure based on a cleanup protocol for dried blood spots and a quantitative (q)PCR to screen for a homozygous deletion of exon 7 of the survival of motor neuron 1 gene (SMN1) that is responsible for >95% of SMA patients. Performance of this setup is evaluated in detail and tested on routine samples. Our cleanup method for nucleic acids from dried blood spots yields enough DNA for diverse subsequent qPCR applications. To date, we have applied this approach to test 213,279 samples within 18 months. Thirty patients were identified and confirmed, implying an incidence of 1:7109 for the homozygous deletion. Using our cleanup method, a rapid workflow could be established to prepare nucleic acids from dried blood spot cards. Targeting the exon 7 deletion, no invalid, false-positive, or false-negative results were reported to date. This allows timely identification of the disease and grants access to the recently introduced treatment options, in most cases before the onset of symptoms. Carriers are not identified, thus, there are no concerns of whether to report them.

Although the value of newborn screening (NBS) for early detection and treatment opportunity in SMA patients is generally accepted, there is still an ongoing discussion about the best strategy in children with 4 and more copies of the SMN2 gene. This gene is known to be the most important but not the only disease modifier.In our SMA-NBS pilot project in Germany comprising 278,970 infants screened between January 2018 and November 2019 were 38 positive cases with a homozygous SMN1 deletion. 40% of them had 4 or more SMN2 copies. The incidence for homozygous SMN1 deletion was 1 : 7350, which is within the known range of SMA incidence in Germany.Of the 15 SMA children with 4 SMN2 copies, one child developed physical signs of SMA by the age of 8 months. Reanalysis of the SMN2 copy number by a different test method revealed 3 copies. Two children had affected siblings with SMA Type III, who were diagnosed only after detection of the index patient in the NBS. One had a positive family history with an affected aunt (onset of disease at the age of 3 years). Three families were lost to medical follow up; two because of socioeconomic reasons and one to avoid the psychological stress associated with the appointments.Decisions on how to handle patients with 4 SMN2 copies are discussed in the light of the experience gathered from our NBS pilot SMA program.

Background: Newborn screening (NBS) programs for treatable metabolic disorders have been enormously successful, but molecular-based screening has not been broadly implemented so far., Methods: This prospective pilot study was performed within the German NBS framework. DNA, extracted from dried blood cards was collected as part of the regular NBS program. As cystinosis has a prevalence of only 1:100,000-1:200,000, a molecular genetic assay for detection of the SMN1 gene mutation with a higher prevalence was also included in the screening process, a genetic defect that leads to spinal muscular atrophy (SMA). First tier multiplex PCR was employed for both diseases. The cystinosis screening employed assays for the three most common CTNS mutations covering 75% of German patients; in case of heterozygosity for one of these mutations, samples were screened by next generation sequencing (NGS) of the CTNS exons for 101 CTNS mutations. A detection rate of 98.5% is predicted using this approach., Results: Between January 15, 2018 and May 31, 2019, 257,734 newborns were screened in Germany for cystinosis. One neonate was diagnosed with cystinosis, consistent with the known incidence of the disease. No false positive or false negatives were detected so far. Screening, communication of findings to parents, and confirmation of diagnosis were accomplished in a multi-disciplinary setting. This program was accomplished with the cooperation of hospitals, physicians, and parents. In the neonate diagnosed with cystinosis, oral cysteamine treatment began on day 18. After 16 months of treatment the child has no clinical signs of renal tubular Fanconi syndrome., Conclusions: This pilot study demonstrates the efficacy of a molecular-based neonatal screening program for cystinosis using an existing national screening framework., (© 2019 The Authors.)

Objective: Spinal muscular atrophy (SMA) is the most common neurodegenerative disease in childhood. The study was conducted to assess the impact of early detection of SMA by newborn screening (NBS) on the clinical course of the disease., Methods: Screening was performed in two federal states of Germany, Bavaria and North Rhine Westphalia, between January 2018 and February 2019. The incidence in the screening population was calculated as number of detected patients with a homozygous deletion in the SMN1-gene per number of screened patients. To get an idea about the incidence of newly diagnosed SMA in the year prior to screening a survey covering all neuropediatric centers in the state of Bavaria was conducted, identifying all SMA-cases in 2017 and 2018. Following positive NBS and confirmatory diagnostic test, treatment was advised according to the recommendations of the "American SMA NBS Multidisciplinary Working Group". Immediate treatment with Nusinersen was recommended in children with 2 and 3 SMN2 copies and a conservative strict follow-up strategy in children with ≥4 copies. All children underwent regular standardized neuropediatric examination, CHOP INTEND and HINE-2 testing as well as electrophysiological exams every 2-3 months., Results: 165,525 children were screened. 22 cases of SMA were identified, meaning an incidence rate of 1:7524. SMN2 copy number analysis showed 2 SMN2 copies in 45% of patients, 3 SMN2 copies in 19 % and 4 SMN2 copies in 36%. These findings are confirmed in the most recent statistical data-cut from 31st August 2019 (incidence 1:7089, 2 SMN2 copies in 44%, 3 in 15% and 4 in 38%). Comparison with up-to-date German data on SMA incidence and the Bavarian survey give evidence that NBS did not lead to a relevant increase in incidence. 10 patients with 2 or 3 SMN2 copies were treated with Nusinersen, starting between 15- 39 days after birth, in 7/10 patients before onset of symptoms. Presymptomatically treated patients (age at last examination: 1- 12 months, median 8 months) showed no muscle weakness by the age of one month to one year. One child with 4 SMN2 copies became symptomatic at the age of 8 months., Conclusions: Newborn screening, resulting in presymptomatic treatment, improves outcome in children with genetically proven SMA. Newborn screening for SMA should be introduced in all countries where therapy is available. An immediate therapy in cases with 4 SMN2 copies should be considered.

Sarcopenia is the age-related loss of muscle mass and function, reducing force generation and mobility in the elderlies. Contributing factors include a severe decrease in both myofiber size and number as well as a decrease in the number of motor neurons innervating muscle fibers (mainly of fast type) which is sometimes accompanied by reinnervation of surviving slow type motor neurons (motor unit remodeling). Reduced mobility and functional limitations characterizing aging can promote a more sedentary lifestyle for older individuals, leading to a vicious circle further worsening muscle performance and the patients' quality of life, predisposing them to an increased risk of disability, and mortality. Several longitudinal studies have shown that regular exercise may extend life expectancy and reduce morbidity in aging people. Based on these findings, the Interreg IVa project aimed to recruit sedentary seniors with a normal life style and to train them for 9 weeks with either leg press (LP) exercise or electrical stimulation (ES). Before and at the end of both training periods, all the subjects were submitted to mobility functional tests and muscle biopsies from the Vastus Lateralis muscles of both legs. No signs of muscle damage and/or of inflammation were observed in muscle biopsies after the training. Functional tests showed that both LP and ES induced improvements of force and mobility of the trained subjects. Morphometrical and immunofluorescent analyses performed on muscle biopsies showed that ES significantly increased the size of fast type muscle fibers (p<0.001), together with a significant increase in the number of Pax7 and NCAM positive satellite cells (p<0.005). A significant decrease of slow type fiber diameter was observed in both ES and LP trained subjects (p<0.001). Altogether these results demonstrate the effectiveness of physical exercise either voluntary (LP) or passive (ES) to improve the functional performances of aging muscles. Here ES is demonstrated to be a safe home-based method to counteract fast type fiber atrophy, typically associated with aging skeletal muscle.