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.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1

Multiplex PCR for simultaneously detection of CTNS variants. a Amplification signal for 57 kb deletion, detection filter 533-610, three samples show an exponential amplification signal, demonstrating the presence of the deletion. One sample was negative in this assay and the last one represents the negative control, both without amplification signal. No differentiation between homo- or heterozygous state is possible. b Amplification of parts of exon 3 used for differentiation between a homo- or heterozygous 57 kb deletion as well as amplification control (Channel 533–610). One sample gives an exponential amplification signal, indicating the presence of the wildtype region used for differentiation, two samples were negative and one is the negative control. c Detection of rs786204501 (c.18_21del GACT) via melting analysis (Channel 498–640). The mutated allele gives a melting peak of app. 55 °C, whereas the wildtype allele shows a peak of app. 46 °C. One sample is heterozygous for this variant, and the two other samples show the wildtype allele. d Detection of rs786204420 (c.926dupG) via melting analysis (Channel 465–510). Wildtype allele gives a melting peak at app. 65 °C and the insertion at app. 72 °C. Two samples are wildtype for the tested variant, with a melting point at 65 °C, one sample is homozygous for the c.926dupG giving a melting peak at 72 °C and one sample is heterozygous for this variant

Fig. 2

Results after 19 months of CTNS screening. Within 19 months of screening, 291, 905 samples were analyzed. 291,023 (99.7%) were negative for any of the three variants in the qPCR assay and reported immediately. 656 (0.22%) samples were positive for a heterozygous 57 kb deletion, 84 (0.028%) showed inconclusive results and were further analyzed by sequencing. 81 (0.027%) and 59 (0.02%) samples were heterozygous for c.18_21del GACT or c.926dupG and sequenced as well. Three potential cystinosis patients could be identified. Two with a homozygous deletion of the 57 kb region, the other sample compound heterozygous 57 kb del with a second variant, previously described as disease causing. All others were reported without pathological findings

Fig. 3

Shift of melting peaks. a Melting analysis to detect rs786204501 (c.18_21del GACT) in the channel 498–640 with a heterozygous sample showing a lowered melting peak (44 °C) next to the wildtype peak (46 °C). Sequencing identified the c.12T>C variant, described as non-pathogenic. The two other samples showed a wildtype and a heterozygous melting peak. b Melting analysis to detect rs786204420 (c.926dupG) in the channel 465–510 with a heterozygous sample showing a lowered melting peak (62 °C) next to the wildtype (65 °C). Sequencing identified the c.922G>A variant, described as disease-causing in HGMD® database. One sample is wildtype for the tested variant, with a melting point at 65 °C, one sample is homozygous for the c.926dupG giving a melting peak at 72 °C and one sample is heterozygous for this variant. c Melting analysis to detect rs786204501 (c.18_21del GACT) in the channel 498–640 with a heterozygous sample showing a lower and more wide melting peak (45 °C) relative to the wildtype peak (46 °C). Sequencing identified the c.29T>C variant, described as disease-causing in HGMD® database. The two other samples showed a wildtype and a heterozygous melting peak

Fig. 4

Time frame of cystinosis screening. Up to 2,000 samples can be processed with this approach, allowing reporting results for 99.7% of all samples in one day. Homo- or compound heterozygous status for the three variations screened for, are confirmed by retesting. Second-tier diagnostic by next-generation sequencing (NGS) provides results within six days for the oldest samples