Expanded Screening of One Million Swedish Babies with R4S and CLIR for Post-Analytical Evaluation of Data.

Sweden has one neonatal screening laboratory, receiving 115 to 120 thousand samples per year. Among the one million babies screened by tandem mass spectrometry from November 2010 until July 2019, a total of 665 babies were recalled and 311 verified as having one of the diseases screened for with this methodology, giving a positive predictive value (PPV) of 47% and an incidence of 1:3200. The PPV was high (41%) already in the first year after start of screening, thanks to the availability of the collaborative project Region 4 Stork database. The PPV is presently 58%. This improvement was achieved by the implementation of second-tier analyses in the screening for methylmalonic aciduria, propionic aciduria, isovaleric aciduria, and homocystinuria, and the employment of various post analytical tools of the Region 4 Stork, and its successor the collaborative laboratory integrated reports.

Heterogeneity of disease-causing variants in the Swedish galactosemia population: Identification of 16 novel GALT variants.

The aim was to determine disease-causing variants in the GALT gene which codes for the enzyme galactose-1-phosphate uridylyltransferase. Loss of activity of this enzyme causes classical galactosemia-a life threatening, treatable disorder, included in the Swedish newborn screening program since 1967. A total of 66 patients with the disease are known in Sweden and 56 index patients were investigated. An additional two patients with Duarte galactosemia were included. The disease-causing variants were identified in all patients. As reported from other countries only a few variants frequently recur in severe disease. The two variants p.(Gln188Arg) (c.563A>G) and p.(Met142Lys) (c.425T>A) are present in several index patients whereas the remaining are found in one to three patients each. The most common variant, p.(Gln188Arg), has an allele frequency of 51% in the cohort. A total of 16 novel variants were found among the 33 different variants in the cohort. Two of these are synonymous variants affecting splicing, demonstrating the importance of the evaluation of synonymous variants at the cDNA level. Concise sentence: Galactosemia is a rare disease in Sweden and the disease-causing variants are heterogenous including two synonymous variants.

Incidence of Glucose-6-Phosphate Dehydrogenase Deficiency among Swedish Newborn Infants.

Sweden has 10.2 million inhabitants and more than 2.4 million have a foreign background. A substantial number of immigrants come from countries where glucose-6-phosphate dehydrogenase deficiency (G6PDD) is frequent. The total birth rate annually in Sweden is approximately 117,000 and newborn screening is centralized to one laboratory. We determined glucose-6-phosphate dehydrogenase (G6PD) activity in 10,098 dried blood spot samples (DBS) from the whole country with a fluorometric assay (LabSystems Diagnostics Oy, Finland). The first 5451 samples were anonymised and run as singletons, whilst the following 4647 samples were coded. Enzyme activity ≤40% of the mean of the day was found in 58 samples (1/170) and among these, 29 had activities ≤10% (1/350). Twenty-nine samples with residual activities between 2-39% in the coded cohort were subjected to Sanger sequencing. Disease-causing variants were identified in 26 out of 29 infants, of which six were girls. In three patients, we did not find any disease-causing variants, although two patients were hemizygous for the known polymorphisms c.1311T>C and c.1365-13C>T. The most common disease-causing variant found in 15 of the 29 samples (12 hemizygotes, two heterozygotes, one homozygote) was the Mediterranean mutation, c.563C>T (p.(Ser188Phe)) in exon 6. G6PDD is thus a surprisingly prevalent disorder in Sweden.

The Spectrum of PAH Mutations and Increase of Milder Forms of Phenylketonuria in Sweden During 1965-2014.

Newborn screening (NBS) for phenylketonuria (PKU) which has a continuum of disease severities has been performed for more than 50 years. The screening method has undergone a continuous development with not only improvements of the positive predictive value but also identification of milder forms of the disease. With the introduction of genetic testing the confirmation of the diagnosis has improved. The Swedish NBS is centralized to one laboratory, which also performs confirmatory testing.Here we present the results of NBS for PKU in Sweden during 1965-2014 describing an increase in diagnosed patients and a shift in the spectrum of phenylalanine hydroxylase (PAH) mutations towards an increasing heterogeneity. Milder mutations common in southern Europe and the Middle East together with lowering of the recall level for phenylalanine (Phe) have led to a shift towards milder phenotypes among the patients identified by the screening program. The inclusion of a Phe and tyrosine (Tyr) ratio as an additional marker has improved the positive predictive value to the present 0.92. Also discussed is what impact earlier sampling has had on the prediction of disease severity, concluding that the shift of age at sampling from 72 to 48 h does not increase the risk of missing patients in need of treatment.

Newborn Screening for Severe Primary Immunodeficiency Diseases in Sweden-a 2-Year Pilot TREC and KREC Screening Study.

Newborn screening for severe primary immunodeficiencies (PID), characterized by T and/or B cell lymphopenia, was carried out in a pilot program in the Stockholm County, Sweden, over a 2-year period, encompassing 58,834 children. T cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) were measured simultaneously using a quantitative PCR-based method on DNA extracted from dried blood spots (DBS), with beta-actin serving as a quality control for DNA quantity. Diagnostic cutoff levels enabling identification of newborns with milder and reversible T and/or B cell lymphopenia were also evaluated. Sixty-four children were recalled for follow-up due to low TREC and/or KREC levels, and three patients with immunodeficiency (Artemis-SCID, ATM, and an as yet unclassified T cell lymphopenia/hypogammaglobulinemia) were identified. Of the positive samples, 24 were associated with prematurity. Thirteen children born to mothers treated with immunosuppressive agents during pregnancy (azathioprine (n = 9), mercaptopurine (n = 1), azathioprine and tacrolimus (n = 3)) showed low KREC levels at birth, which spontaneously normalized. Twenty-nine newborns had no apparent cause identified for their abnormal results, but normalized with time. Children with trisomy 21 (n = 43) showed a lower median number of both TREC (104 vs. 174 copies/µL blood) and KREC (45 vs. 100 copies/3.2 mm blood spot), but only one, born prematurely, fell below the cutoff level. Two children diagnosed with DiGeorge syndrome were found to have low TREC levels, but these were still above the cutoff level. This is the first large-scale screening study with a simultaneous detection of both TREC and KREC, allowing identification of newborns with both T and B cell defects.

Galactosemia screening with low false-positive recall rate: the Swedish experience.

Newborn screening was implemented in the 1960s with screening for phenylketonuria (PKU). In the same decade, it became possible to screen for classical galactosemia, a rare autosomal recessive inherited disorder, which is potentially life threatening if not treated. While newborn screening for PKU has become almost universal, galactosemia is included only in a minority of European newborn screening programs. The major arguments why galactosemia is excluded from newborn screening programs are that the disease can be diagnosed clinically, there is a high rate of false positives and long-term complications are common despite early diagnosis.Here, we report how we have decreased the number of false-positive galactosemia recalls to less than 0.01%, using a two-tier test strategy. All samples are tested with the Beutler blood spot test, a method that measures galactose-1-phosphate uridyltransferase activity. Samples with less than ≤15% activity are tested for galactose with a galactose dehydrogenase test (the rapid GAL-DH test), which catalyzes the oxidation of galactose and the reduction of NAD(+) to NADH that is estimated visually by fluorescence under UV-light. Both tests are semiquantitative.With this strategy, screening for galactosemia is inexpensive, does not demand a heavy workload, and has a low false-positive re-call rate. The incidence of classical galactosemia in Sweden is 1/100,000, which is lower than the reported incidence in other European countries. Despite this, newborn screening for galactosemia has never been questioned. Concise sentence: Screening for galactosemia using well-established methods to reduce the false-positive rate.

A novel mucopolysaccharidosis type I associated splice site mutation and IDUA splice variants.

Mucopolysaccharidosis type I is an autosomal recessive disorder caused by deficiency of α-l-iduronidase, encoded by the IDUA gene. More than 100 disease causing mutations have been reported in the gene, resulting in a wide range of phenotypes. Here we describe a previously unreported IDUA splice site mutation (NG_008103.1:g.21632G>C; NM_000203.3:c.1727+3G>C) causing a Hurler phenotype in a patient heterozygous for the common p.Q70X (NG_008103.1:g.5862C>T) mutation. Sequence analysis of IDUA transcripts demonstrated that the g.21632G>C mutation results in aberrant splicing of intron 12 (NM_000203.3:c.1727_1728insGTCC), introducing a frame shift and premature termination codon (NP_000194.2:p.Cys577SerfsX15). Gene expression studies suggest that the deleterious effect of the mutation is primarily due to a C-terminal truncation of the encoded polypeptide. Furthermore, we observed that both normal and mutant IDUA alleles give rise to alternatively spliced transcripts in leukocytes. Exclusion of exon 4 appeared to be the predominant alternative splicing event, probably resulting in polypeptides lacking iduronidase activity. The Hurler patient demonstrated exon 4 skipping in 5.6% of IDUA transcripts, while exon 4 skipping ranged 25-34% of transcripts among healthy individuals (n=5). Alternative splicing might represent a mechanism for regulation of this enzyme, and the lower level of exon 4 skipping in the patient might be a response to intracellular accumulation of iduronidase substrates. Molecular characterization of IDUA mutations and splicing may assist early prediction of mucopolysaccharidosis type I phenotypes and increase the understanding of disease mechanisms. This is important considering the choice of current treatment options and for the development of future therapies.

Profound biotinidase deficiency: a rare disease among native Swedes.

Biotinidase deficiency is an autosomal recessive metabolic disorder included in many newborn screening programmes. Prior to the introduction of screening for biotinidase deficiency in Sweden in 2002, the disorder was almost unknown, with only one case diagnosed clinically. Biotinidase activity was measured in dried blood spots with a semiquantitative method using biotin-6-amidoquinoline as substrate. The cutoff value was set at 25% (later lowered to 20%) of the mean activity of all samples measured on that day. The disorder was confirmed by quantitative determination of biotinidase activity in plasma and DNA analyses. Over a period of 6 years, 13 patients were identified among 637,452 screened newborns and 5,068 adoptive/immigrant children. None of the patients had clinical symptoms at the time of diagnosis. Six patients had profound biotinidase deficiency, with an activity of 0-5% of normal in plasma. Four of these patients were born to parents who were first cousins of Middle Eastern or African origin. Eighteen gene alterations were identified, nine of which have not previously been described: seven mutations p.L83S (c.248T > C), p.R148H (c.443G > A), p.N202I (c.605A > T), p.I255T (c.764T > C), p.N402S (c.1205A > G), p.L405P (c.1214T > C), p.G445R (c.1333G > A) and two silent mutations p.L71L (c.211C > T) and p.L215L (c.645C > T). The predicted severity of the novel mutations was analyzed by sorting intolerant from tolerant (SIFT) and polymorphism phenotyping (PolyPhen), predicting p.L83S, p.L405P and p.G445R as severe mutations. Due to the high rate of immigrants since 1990 from non-Nordic countries, the incidence of biotinidase deficiency is similar to that found in many other Western countries.