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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.

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.

Background: Spinal muscular atrophy (SMA) is the most common neurodegenerative disease in childhood. Since early diagnosis and treatment are essential to prevent major disability, newborn screening (NBS) has come into focus. Methods: The pilot project in two federal states of Germany started in January 2018 and is still ongoing. Genetic screening via PCR of the SMN1 gene from dried blood spots was implemented in the routine NBS structure. Follow-up included neuropediatric and neurophysiological examinations, CHOP INTEND and HINE-2. Findings: Among 297,163 screened children, 43 cases of SMA were identified resulting in an incidence of 1:6910. Two SMN2 copies were identified in 39.5%, 3 SMN2 copies in 21% and 4 SMN2 copies in 39.5% of patients. In 21 patients with ≤3 SMN2 copies, treatment with Nusinersen was started within 14-39 days after birth. To date, all presymptomatically treated patients have remained asymptomatic. 41% of patients with 2 SMN2 copies had already had early, mostly subtle signs of disease such as ulnar CMAP

A rapid and easily workable alternative (MEP: Munich Extraction Protocol) is outlined to our current RNA purification method for circumstances where there is limited availability of test kits. Details are available upon request.

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. Keywords: Cystinosis, Molecular-based newborn screening, Efficacy, Follow-up