Genetic Newborn Screening for Cystinosis and Primary Hyperoxaluria

Background and aims. Population-based newborn screening (NBS) is an important public health program that has vastly improved the course of several diseases through early detection. The selection of screened disorders generally follows the 10 principles outlined by Wilson and Jungner. In Germany, NBS has been a voluntary National Health Service program since 1969 which currently covers 17 disorders. Current NBS methods, which employ tandem mass-spectrometric analysis of newborn dried blood spots, cannot detect many potentially treatable genetic conditions. At the same time, molecular-based NBS is increasingly feasible because DNA can be extracted from a dried blood spot, next generation sequencing has become economical, and molecular diagnostics have greater reliability and increased validity as genetic databases become more refined and comprehensive. Nephropathic cystinosis and hyperoxaluria (PH) are eligible for molecular-based NBS because effective therapies are available.

In a first pilot project, the scientific basis for NBS for cystinosis could already be established. The aim of this study is to demonstrate the transferability of genetic NBS for Cystinosis to other laboratories and to lay the scientific basis for screening for PH.

Specifically, the study will investigate whether the inclusion of these diseases into general NBS should be recommended. By observing the identified infants in comparison to patients symptomatically diagnosed outside of the pilot project, it will be determined whether and to what extent early diagnosis and therapy lead to a more favorable prognosis.

Cystinosis Nephropathic cystinosis, due to impaired transport of cystine out of lysosomes, occurs with an incidence of 1 in 100-200,000 live births. It is characterized by renal Fanconi syndrome in the first year of life and glomerular dysfunction progression to end-stage kidney disease by approximately 10 years of age. Treatment with oral cysteamine therapy helps preserve glomerular function, but affected individuals eventually require kidney replacement therapy Cysteamine treatment generally begins at the time of diagnosis in the second year of life, but some glomerular and tubular damage has already occurred by then. This situation could be ameliorated by diagnosing patients shortly after birth, employing molecular genetics-based newborn screening. Standard mass spectrometry-based methods for newborn screening cannot detect the increased cystine content of cystinosis leukocytes.

For cystinosis screening, the first tier involved multiplex PCR to detect two of the three most common CTNS mutations in Germany. Heterozygous samples will be submitted to amplicon-based next-generation sequencing for 175 pathogenic CTNS mutations (Labor Limbach, Mainz). A detection rate of 96.5% is predicted using this approach.

Primary Hyperoxaluria Three different defaults in the glyoxylate metabolism lead to PH. The severe type PH1 is the most common variant (1-3 out of 106 patients). Population-based studies estimate a prevalence of 1:58,000. The estimated number of unreported cases is high. The deposit of calcium oxalate crystals in the kidneys triggers a chronic inflammation which results in terminal renal failure. Decreased oxalate excretion in the urine leads to high oxalate concentrations in the plasma and subsequently deposits of calcium oxalate in the organs and tissue (systemic oxalosis). The clinical course is highly variable. Examples of infantile oxalosis with early renal function loss up to a-/oligosymptomatic patients in adulthood are described.

Clinically, PH2 is calmer, but about 50% of patients develop end-stage renal failure.

PH3 used to be considered a mild variant, but it is known today that PH3 patients can also develop kidney stones in childhood and develop terminal renal failure with a systemic oxalosis.

The PH registry of OxalEurope (European hyperoxaluria consortium) currently lists 1137 genetically diagnosed patients, comprising 81.9% with PH1, 9.8% with PH2 and 8.4% with PH3. In the molecular genetic evaluation of the German registry, the results are: 74.1% PH1, 7.9% PH2 and 17% PH3.

So far, primary hyperoxaluria has been diagnosed according to clinical findings through urine analysis (or plasma test in case of terminal renal failure). Therefore, the diagnosis is usually made once patients have already developed terminal renal failure. But existing medications (Vitamin B6) or the new RNAi medications (Lumasiran, Nedosiran) can prevent renal failure and are making the disease treatable. The purpose of this pilot project is to identify the most common mutations in the AGXT gene and in the HOGA1 gene.

Workflow Study population Hospitals in Germany are free to choose between 11 certified laboratories for NBS. In this project, the Screening-Laboratory Hannover informs its senders about the possibility to extend the routinely established NBS by the genetic screening for cystinosis and PH.

The study population includes newborns whose parents wish to participate in the pilot project and have provided written informed consent.

Parent information and consent Before screening is performed, the newborn's parents are comprehensively informed about the possibility of screening for the various diseases by the physician responsible for NBS (gynecologist, pediatrician). An additional information and consent sheet for cystinosis and PH screening is inserted into the information brochure already available for regular NBS. Consent must be documented with the signature of at least one parent and the signature of the informing physician on the consent form. The consent form for the pilot project also includes consent for the transfer of contact data and findings to a specialized center in the event of an abnormal screening result. The laboratory must receive the consent form for the project prior to analysis. Results that turn out normal are reported to the responsible submitting physician according to the NBS guidelines for children. The sender must check whether there is a result for each blood sample taken for screening (result return control).

In case of a positive result, the laboratory must first inform the sender and clarify whether the child is still hospitalized. In that case, the sender obtains the initial information from the parents and notifies an expert for the respective diagnosis (Cystinosis: Priv.-Doz. Dr. med.

1. Hohenfellner, Rosenheim; Hyperoxaluria: Prof. Dr. med. B. Hoppe, Bonn). If the child has already been discharged from hospital, the laboratory will contact the relevant expert directly which is covered by the informed consent.

Sampling The molecular genetic screening is performed using the same dried blood spot card as the routine NBS.

In general, for all invalid results (i.e., if the control reaction fails) as well as all abnormal results (Cystinosis: homozygous or heterozygous for the 57-kb deletion and /or c.18_21delGACT, p.T7Ffs*7 ; PH1, c.508G>A, PH3, C700+5G>T, homozygous and/or heterozygous) the test will be internally repeated for confirmation using the existing blood sample.

Measurements and methods A real-time quadruplex PCR is performed for the four mutations described above. Cystinosis: Detection is carried out by detection of the binding or by melting analysis of fluorescence-labeled probes. In samples with a positive result due to heterozygosity mutations, the exons of the cystinosin gene (CTNS) are sequenced using next generation sequencing (NBS) in an amplicon-based method. This ensures that only the desired exons and no other DNA areas are examined. DNA already extracted for real-time multiplex PCR can be used for NBS. With NEBNext Direct Genotyping Solution all analyzable areas of CTNS gene are covered (partial overlapping), which preserve a completely sequence information of all exons and specific introns. Therefore, the DNA will be enzymatically fragmented and then ligated with illuminable adaptors. The adaptors contain individual indices. Afterwards the biotin beads will be used for the target enrichment. They will be augmented with streptavidin beads. Afterwards off-target sequences will be removed, and the libraries will be amplified by PCR. The actual sequencing is carried out using a device from Illumina (MiniSeq). The sequences are evaluated using a software that performs an automatic comparison with a reference sequence, thereby detecting mutations.

PH: The detection will be done through heat analysis with fluorescent tagged leads. In case of positive results (also due to heterozygotic constellations) a clarification is planned in collaboration with the German hyperoxaluria center. Therefore, spot urine will be tested for oxalate and further parameters in the glyoxylate metabolism.

Examination results Cystinosis: If the most frequent mutations of CTNS are detected as homozygous or compound heterozygous mutations, the cystinosis screening is considered positive. Using next-generation sequencing, an additional 175 mutations recorded in the literature will be detected. In all other cases, even those involving heterozygous status, cystinosis screening is normal.

PH: The hyperoxaluria screening is positive, if PH1 (AGXT-Gen:c508G>A) and/or PH3 (HOGA-gene: c.700+5G>T) is homozygote detected. In heterozygote genetic carrier with positive screening, a subsequent urine test performed in the Wisplinghoff laboratory in Cologne (Köln) will clarify the finding.

Detection rate, false positive and false negative results An overall detection rate of above 95% is assumed for both diseases. False negative screening results are unlikely.

For cystinosis, false negative results due to previously unknown rare mutations or patients carrying neither of the two most frequent mutations are possible.

False positive screening results are also very unlikely. With cystinosis, a heterozygous sample due to "allelic dropout" (PCR failure of an allele due to mutations in the primer binding region) may incorrectly appear homozygous.

In PH, the disease is not excluded in case of heterozygous mutation, further diagnostics is necessary and planned.

In case of corresponding symptoms, cystinosis or PH must be included in the differential diagnosis, which usually takes place in one of the few treatment centers. Such cases can only be documented in the course of time via feedback of the attending centers can be documented.

Confirmation of the diagnosis Cystinosis: In patients with either a homozygous or compound heterozygous mutation in the CTNS-gene, the diagnosis will be confirmed by determining the intraleukocytic cystine level from 2,3 ml EDTA blood. This sample will be sent to the metabolic laboratory in Heidelberg within the first 14 days of life.

PH: In homozygous carriers of the mutations in the PH1 or PH3 genes, a control examination of spot urine or of plasma will follow, which will be carried out in the German hyperoxaluria Centre.

The results of the verification tests will be transferred to the screening laboratory for quality control.

Caring for affected children Cystinosis: Children with positive cystinosis screening results and their parents are referred to the nearest center for metabolic diseases. Therapy with cysteamine can be started immediately after confirmation of the diagnosis. Parents are informed about the possibility of an interdisciplinary cystinosis consultation in Rosenheim.

PH: Children with a positive screening result will be primarily referred to the German hyperoxaluria center in Bonn.

Further treatment of these patients is performed at the nearest hospital of their choice.

Project size For financial reasons, the project is limited to 200,000 samples, with the possibility of expansion.