Cystinosis in Newborn Screening

Cystinosis in Newborn Screening: Comparison

Please note this is a comparison between Version 1 by Katharina Hohenfellner and Version 4 by Amina Yu.

Newborn screening programmes (NBS) aism to identify presymptomatic newborns with rare serious or fatal disorders that can be successfully treated, thereby achieving a significant reduction in morbidity and mortality. Infantile nephropathic cystinosis (INC) is a very rare lysosomal metabolic disorder. With the introduction of cysteamine therapy in the early 1980s and the possibility of renal replacement therapy in infancy, patients with cystinosis can now reach adulthood. Early diagnosis of cystinosis remains important as this enables initiation of cysteamine at the earliest opportunity to support renal and patient newborn screening; infantile nephropathic cystinosis; clinical course; CTNS-pathogenic variants; newborn screening for cystinosisurvival.

newborn screening
infantile nephropathic cystinosis
newborn screening for cystinosis

1. Cystinosis

Cystinosis is a rare autosomal recessive systemic disease with high morbidity and mortality caused by pathogenic variants in the CTNS gene that encodes the lysosomal cystine transporter cystinosin, leading to accumulation of cystine within the lysosome ^[1][2]. Life-long cystine-depleting therapy with oral cysteamine, the only specific therapy for cystinosis, along with the availability of renal replacement therapy in childhood, has dramatically improved patient outcomes ^[3]. There is robust evidence that early initiation and sustained therapy with cysteamine are both essential for delaying progression to chronic kidney disease (CKD) and end-organ damage ^[4].

Cystinosis is a rare autosomal recessive systemic disease with high morbidity and mortality caused by pathogenic variants in the CTNS gene that encodes the lysosomal cystine transporter cystinosin, leading to accumulation of cystine within the lysosome [38,39]. Life-long cystine-depleting therapy with oral cysteamine, the only specific therapy for cystinosis, along with the availability of renal replacement therapy in childhood, has dramatically improved patient outcomes [40]. There is robust evidence that early initiation and sustained therapy with cysteamine are both essential for delaying progression to chronic kidney disease (CKD) and end-organ damage [41].

Infantile nephropathic cystinosis is the most common cause of renal Fanconi syndrome in children and is a hallmark of the disease ^[5]. Renal Fanconi syndrome presents with proximal renal tubular acidosis along with a generalized dysfunction of the proximal tubule, characterized by the presence of polyuria, glycosuria, phosphaturia, tubular proteinuria, growth retardation, and rickets; later glomerular involvement leads to progressive kidney failure. The proximal tubular cells (PTCs) are first to be affected ^[6]. However, evidence from mouse models suggests that differentiation (structural changes) of PTCs starts prior to the accumulation of cystine crystals in both PTCs and the interstitium, leading to a loss of their brush border, flattening and thickening of tubular basement membrane, and the eventual development of the characteristic swan-neck deformity ^[7]. These changes progress to tubular atrophy and, in addition, heavy inflammatory cell infiltrates can be observed in the renal interstitium. Glomerular involvement with multinucleated podocytes and focal segmental glomerulosclerosis lesions can be seen in renal biopsies ^[5]. While the defect in cystine transport by cystinosin is the hallmark of cystinosis, it is not the only key player in the pathogenesis of renal Fanconi syndrome; cystinosin has additional roles, including regulation of autophagy, mTOR signalling, lysosomal biogenesis, and vesicle trafficking in proximal tubular epithelial cells.

Infantile nephropathic cystinosis is the most common cause of renal Fanconi syndrome in children and is a hallmark of the disease [42]. Renal Fanconi syndrome presents with proximal renal tubular acidosis along with a generalized dysfunction of the proximal tubule, characterized by the presence of polyuria, glycosuria, phosphaturia, tubular proteinuria, growth retardation, and rickets; later glomerular involvement leads to progressive kidney failure. The proximal tubular cells (PTCs) are first to be affected [43]. However, evidence from mouse models suggests that differentiation (structural changes) of PTCs starts prior to the accumulation of cystine crystals in both PTCs and the interstitium, leading to a loss of their brush border, flattening and thickening of tubular basement membrane, and the eventual development of the characteristic swan-neck deformity [44]. These changes progress to tubular atrophy and, in addition, heavy inflammatory cell infiltrates can be observed in the renal interstitium. Glomerular involvement with multinucleated podocytes and focal segmental glomerulosclerosis lesions can be seen in renal biopsies [42]. While the defect in cystine transport by cystinosin is the hallmark of cystinosis, it is not the only key player in the pathogenesis of renal Fanconi syndrome; cystinosin has additional roles, including regulation of autophagy, mTOR signalling, lysosomal biogenesis, and vesicle trafficking in proximal tubular epithelial cells.

Early diagnosis of cystinosis enables treatment with cysteamine, the only specific therapy for the disease, which should be administered as early as possible and continued throughout the life of the patient ^[1]. It is well accepted that early treatment with cysteamine improves patient outcome, delays progression to renal failure and prevents or attenuates end-organ damage ^{[4][8][9][10][11][12]}. Initiation of cysteamine before 3, 2.5, or even <2 years of age has been associated with preservation of renal function in patients with cystinosis ^{[3][8][9][10][13][14][15]}; thus, patient age at initiation of cysteamine therapy appears to be a major predictive factor of renal survival. In a large international contemporary cohort of 453 patients with cystinosis, cysteamine was initiated in 89% of patients at a median age of 1.6 years, and a near linear relationship between the age of cysteamine initiation and renal function was observed ^[10]; patients treated before the age of 1 year exhibited the best renal outcome. All these findings provide the rationale to develop NBS to diagnose cystinosis as early as possible, ideally before the development of clinical manifestations and irreversible PTC damage prior to cystine crystal accumulation.

Early diagnosis of cystinosis enables treatment with cysteamine, the only specific therapy for the disease, which should be administered as early as possible and continued throughout the life of the patient [38]. It is well accepted that early treatment with cysteamine improves patient outcome, delays progression to renal failure and prevents or attenuates end-organ damage [41,45,46,47,48,49]. Initiation of cysteamine before 3, 2.5, or even <2 years of age has been associated with preservation of renal function in patients with cystinosis [40,45,46,47,50,51,52]; thus, patient age at initiation of cysteamine therapy appears to be a major predictive factor of renal survival. In a large international contemporary cohort of 453 patients with cystinosis, cysteamine was initiated in 89% of patients at a median age of 1.6 years, and a near linear relationship between the age of cysteamine initiation and renal function was observed [47]; patients treated before the age of 1 year exhibited the best renal outcome. All these findings provide the rationale to develop NBS to diagnose cystinosis as early as possible, ideally before the development of clinical manifestations and irreversible PTC damage prior to cystine crystal accumulation.

2. Prenatal Testing and Preimplantation Genetic Diagnosis

Families known to be at risk of Infantile nephropathic cystinosis (INC) can consider options such as preimplantation genetic diagnosis or prenatal diagnosis ^[16]. There are two options for prenatal testing: biochemical testing and molecular genetic testing ^[16]. For pregnancies at risk of INC, prenatal diagnosis is possible biochemically, by measuring

2. Prenatal Testing and Preimplantation Genetic Diagnosis

Families known to be at risk of INC can consider options such as preimplantation genetic diagnosis or prenatal diagnosis [59]. There are two options for prenatal testing: biochemical testing and molecular genetic testing [59]. For pregnancies at risk of INC, prenatal diagnosis is possible biochemically, by measuring

³⁵S-labeled cystine accumulation in cultured amniocytes (14–16 weeks of gestation) or chorionic villi samples (CVS) (8–9 weeks of gestation) and by a direct measurement of cystine in uncultured CVS ^[17]. Once the CTNS pathogenic variants have been identified in an affected family member, for a pregnancy at increased risk and preimplantation genetic diagnosis for cystinosis are possible. Differences in perspective may exist among medical professionals and within families regarding the use of prenatal testing, particularly where this is being considered for the purpose of pregnancy termination rather than early diagnosis of a target disease. DNA analysis for detecting mutant alleles is currently the most frequently used antenatal screening method. While most clinical centres typically consider decisions regarding prenatal testing to be the parents’ choice, discussion of any potential issues with the medical team remains important, together with the possible involvement of a genetic counsellor.

3. Newborn Screening for Cystinosis

The implementation of genetic testing in Newborn screening (NBS) was undertaken in Utah in 1998 for the detection of sickle cell disease ^[18]. Molecular biological methods have since been introduced as first tier, second tier, or third tier methods in NBS ^[19]. Currently, the diagnosis of cystinosis is based on the presence of elevated cystine levels in white blood cells ^[2]; this method is unsuitable for NBS. In 2018, as part of a pilot one, the existing German NBS programme was expanded to incorporate high-throughput first-tier molecular genetic screening for cystinosis and spinal muscular atrophy (SMA) ^[20][21]. Both congenital disorders are suitable for molecular-based NBS because they have known genetic causes and effective therapies are available. Based on the results of the pilot project, SMA screening has been successfully included as a regular part of NBS in Germany ^[22]. For cystinosis screening in Germany, the first tier involved multiplex PCR to detect the three most common CTNS pathogenic variants, for example, a 57 kb deletion, c.18_21delGACT, p.T7Ffs*7, and c.926dupG, p.S310Qfs*55 ^[23]. Heterozygous samples were submitted to amplicon-based next-generation sequencing for 101 pathogenic CTNS pathogenic variants published at the time. A detection rate of 98.5% was subsequently predicted using this approach ^[20]. In 299,631 newborns, two patients with a homozygous 57-kb pathogenic variant and one patient with a 57-kb compound homozygous pathogenic variant were identified. A total of 805 patients with heterozygous pathogenic variants were identified, 655 with 57-kb pathogenic variant, 85 with c.18_21delGACT, p.T7Ffs*7, and 65 with c.926dupG, p.S310Qfs*55. In the first patient identified with a homozygous 57-kb pathogenic variant and confirmed diagnosis by determining the leucocyte cystine level (2.82 nmol cystine/mg protein; normal, <0.2), treatment was initiated within the first month of life ^[24]. Even at the age of 3.5 years, the patient presented with normal physical development (height 20% percentile, weight 21% percentile) and without renal Fanconi syndrome or proteinuria. Apart from cysteamine treatment, the patient did not require any other pharmaceutical therapy. Unfortunately, the mother of the second patient with a homozygous 57-kb deletion initially refused NBS for cystinosis. At 8 months of age, the child was admitted to an intensive care unit with a severe electrolytic disturbance. In the course of the latter, a dried blood (DBS) card was re-sent to the screening laboratory and re-evaluated to include cystinosis, which yielded a positive test result. The patient then presented with the full clinical presentation of Fanconi syndrome and required high electrolyte replacement and growth hormone therapy in addition to cysteamine therapy. A third neonate, screened as heterozygous for the common 57-kb deletion, was found to harbour an additional promotor variant (c.-512G>C) in CTNS previously reported as being disease causing. However, according to current ACMG schemes, the respective promotor variant needs to be reclassified as a non-pathogenic change ^[24]. In fact, this infant showed no biochemical evidence of cystinosis, with a normal leucocyte cystine level, i.e., <0.2 nmol cystine/mg protein). Detection rates estimate the known incidence of cystinosis at (1:150,000–1:200,000) ^[25]. False positive and false negative results did not occur until now. One key requirement of all NBS programmes is that they must provide direct clinical benefit ^[26]. Patients with cystinosis are generally diagnosed at 12–18 months of age, by which time significant renal tubular and glomerular damage has already occurred ^[27][28]. As previously described, early treatment with oral cysteamine has salutary effects on preservation of renal function, growth, and prevention of late complications of the disease ^[27]. For those few infants treated shortly after birth due to an older sibling already having cystinosis, even the renal tubular Fanconi syndrome that typically presents in the first months of life was ameliorated in these individuals ^[3]. Molecular screening for the 57-kb pathogenic variant can be combined with an existing NBS for severe combined immunodeficiency (SCID) and/or SMA. According to the results of the German pilot project, 655 of approximately 300,000 newborns carried a heterozygous 57-kb pathogenic variant, which required further screening with NGS for defined CTNS-pathogenic variants ^[24]. The feasibility of implementing NGS in a regular NBS programme has been demonstrated in Norway, where NBS was increased from 2 diseases to 25 in 2018 ^[29], using a second-tier strategy utilizing MS/MS methodologies and NGS for certain diseases. Thus, screening in Northern Europe and North America for the 57-kb pathogenic variant homozygous and heterozygous with downstream NGS, where the 57-kb allele accounts for 50–70% of the alleles, appears to be feasible ^[23][30]; this is further supported by the fact that also in other target diseases not all patients are identified (for example, late-onset hypothyroidism and atypical adrenal hyperplasia). Due to the heterogeneity of screening panels in different countries, the often slow and difficult implementation of additional target diseases, and the limited availability of genomic sequencing in public health and clinical settings, commercial laboratories have begun to offer genomic screening panels for newborns. Hopefully, these will not be as successful as the commercial tests already available from Ancestry, 23andMe, or MyHeritage, whose 2019 databases were estimated at 20 million, 12 million, and 2.5 million, respectively ^[31][32].

S-labeled cystine accumulation in cultured amniocytes (14–16 weeks of gestation) or chorionic villi samples (CVS) (8–9 weeks of gestation) and by a direct measurement of cystine in uncultured CVS [60]. Once the CTNS pathogenic variants have been identified in an affected family member, for a pregnancy at increased risk and preimplantation genetic diagnosis for cystinosis are possible. Differences in perspective may exist among medical professionals and within families regarding the use of prenatal testing, particularly where this is being considered for the purpose of pregnancy termination rather than early diagnosis of a target disease. DNA analysis for detecting mutant alleles is currently the most frequently used antenatal screening method. While most clinical centres typically consider decisions regarding prenatal testing to be the parents’ choice, discussion of any potential issues with the medical team remains important, together with the possible involvement of a genetic counsellor.

3. Newborn Screening for Cystinosis

The implementation of genetic testing in NBS was undertaken in Utah in 1998 for the detection of sickle cell disease [73]. Molecular biological methods have since been introduced as first tier, second tier, or third tier methods in NBS [74].

Currently, the diagnosis of cystinosis is based on the presence of elevated cystine levels in white blood cells [39]; this method is unsuitable for NBS. In 2018, as part of a pilot study, the existing German NBS programme was expanded to incorporate high-throughput first-tier molecular genetic screening for cystinosis and spinal muscular atrophy (SMA) [75,76]. Both congenital disorders are suitable for molecular-based NBS because they have known genetic causes and effective therapies are available. Based on the results of the pilot project, SMA screening has been successfully included as a regular part of NBS in Germany [77].

For cystinosis screening in Germany, the first tier involved multiplex PCR to detect the three most common CTNS pathogenic variants, i.e., a 57 kb deletion, c.18_21delGACT, p.T7Ffs*7, and c.926dupG, p.S310Qfs*55 [64]. Heterozygous samples were submitted to amplicon-based next-generation sequencing for 101 pathogenic CTNS pathogenic variants published at the time. A detection rate of 98.5% was subsequently predicted using this approach [75]. In 299,631 newborns, two patients with a homozygous 57-kb pathogenic variant and one patient with a 57-kb compound homozygous pathogenic variant were identified. A total of 805 patients with heterozygous pathogenic variants were identified, 655 with 57-kb pathogenic variant, 85 with c.18_21delGACT, p.T7Ffs*7, and 65 with c.926dupG, p.S310Qfs*55.

In the first patient identified with a homozygous 57-kb pathogenic variant and confirmed diagnosis by determining the leucocyte cystine level (2.82 nmol cystine/mg protein; normal, <0.2), treatment was initiated within the first month of life [78]. Even at the age of 3.5 years, the patient presented with normal physical development (height 20% percentile, weight 21% percentile) and without renal Fanconi syndrome or proteinuria. Apart from cysteamine treatment, the patient did not require any other pharmaceutical therapy. Unfortunately, the mother of the second patient with a homozygous 57-kb deletion initially refused NBS for cystinosis. At 8 months of age, the child was admitted to an intensive care unit with a severe electrolytic disturbance. In the course of the latter, a dried blood (DBS) card was re-sent to the screening laboratory and re-evaluated to include cystinosis, which yielded a positive test result. The patient then presented with the full clinical presentation of Fanconi syndrome and required high electrolyte replacement and growth hormone therapy in addition to cysteamine therapy. A third neonate, screened as heterozygous for the common 57-kb deletion, was found to harbour an additional promotor variant (c.-512G>C) in CTNS previously reported as being disease causing. However, according to current ACMG schemes, the respective promotor variant needs to be reclassified as a non-pathogenic change [78]. In fact, this infant showed no biochemical evidence of cystinosis, with a normal leucocyte cystine level, i.e., <0.2 nmol cystine/mg protein).

Detection rates estimate the known incidence of cystinosis at (1:150,000–1:200,000) [79]. False positive and false negative results did not occur until now. One key requirement of all NBS programmes is that they must provide direct clinical benefit [21]. Patients with cystinosis are generally diagnosed at 12–18 months of age, by which time significant renal tubular and glomerular damage has already occurred [58,80]. As previously described, early treatment with oral cysteamine has salutary effects on preservation of renal function, growth, and prevention of late complications of the disease [58]. For those few infants treated shortly after birth due to an older sibling already having cystinosis, even the renal tubular Fanconi syndrome that typically presents in the first months of life was ameliorated in these individuals [40].

Molecular screening for the 57-kb pathogenic variant can be combined with an existing NBS for severe combined immunodeficiency (SCID) and/or SMA. According to the results of the German pilot project, 655 of approximately 300,000 newborns carried a heterozygous 57-kb pathogenic variant, which required further screening with NGS for defined CTNS-pathogenic variants [78]. The feasibility of implementing NGS in a regular NBS programme has been demonstrated in Norway, where NBS was increased from 2 diseases to 25 in 2018 [24], using a second-tier strategy utilizing MS/MS methodologies and NGS for certain diseases. Thus, screening in Northern Europe and North America for the 57-kb pathogenic variant homozygous and heterozygous with downstream NGS, where the 57-kb allele accounts for 50–70% of the alleles, appears to be feasible [63,64]; this is further supported by the fact that also in other target diseases not all patients are identified (e.g., late-onset hypothyroidism and atypical adrenal hyperplasia).

Due to the heterogeneity of screening panels in different countries, the often slow and difficult implementation of additional target diseases, and the limited availability of genomic sequencing in public health and clinical settings, commercial laboratories have begun to offer genomic screening panels for newborns. Hopefully, these will not be as successful as the commercial tests already available from Ancestry, 23andMe, or MyHeritage, whose 2019 databases were estimated at 20 million, 12 million, and 2.5 million, respectively [81,82].