Next generation sequencing as second-tier test in high-throughput newborn screening for nephropathic cystinosis

Abstract

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.

An international cohort study spanning five decades assessed outcomes of nephropathic cystinosis

“…To date, these have been tested only in a few studies using a next-generation sequencing approach. 21 Because the Fanconi syndrome associated with NC is characterized by early onset glycosuria and low-molecular weight proteinuria, a urine analysis-based screening would also allow practitioners to suspect the diagnosis very early, although not necessarily at birth. This type of screening in Japan, for example, has been very successful in identifying children with Dent disease.…”

Molecular Mechanisms and Treatment Options of Nephropathic Cystinosis

“…Such screens have long been on the research agenda, but the reliable detection of elevated cystine in blood collected on filter paper for the purposes of newborn screening is technically hampered by oxidation of cytosolic cysteine to cystine. A combination of high-throughput PCR for detecting the three most common mutations followed by next-generation sequencing for identifying mutations on a second allele recently proved successful and allowed for the identification of two patients with cystinosis out of 292 000 newborns [93]. Early treatment immediately after diagnosis may support an improved clinical outcome with respect to RFS development [94].…”

Highlights on Genomics Applications for Lysosomal Storage Diseases

“…On the other hand, genotyping-based NBS seems the better solution for high-risk neonates admitted to the neonatal intensive care unit (NICU), or for those disorders with no existing biochemical marker [ 76 , 77 ]. For example, in the case of nephropathic cystinosis, PCR- and NGS-based analyses have been used for NBS [ 78 ]. Therefore, as outlined by the Newborn Sequencing in Genomic Medicine and Public Health (NSIGHT) consortium, “sequencing technology can be beneficially used in newborns when its use is nuanced and attentive to context” [ 73 ].…”