Neonatal screening for congenital hypothyroidism in Sweden 1980-2013: effects of lowering the thyroid-stimulating hormone threshold.

OBJECTIVE: This study aims to evaluate the neonatal screening for congenital hypothyroidism (CH) and the diagnosis CH in the national health registers and to study the effects of lowering screening thyroid-stimulating hormone (TSH) threshold on the incidence of CH and birth characteristics of screening positive and negative CH children. DESIGN: This is a nationwide register-study of all children (n = 3 427 240) in the Swedish Medical Birth Register (MBR) and national cohort for screening positive infants (n = 1577) in 1980-2013. METHODS: The study population was further linked to several other Swedish health registers. Evaluation of the CH screening and CH diagnosis was performed with levothyroxine use in the first year of life as reference. The incidence of CH was estimated by the Clopper-Pearson method. Regression models were used to study associations between CH and birth characteristics. RESULTS: The neonatal CH screening had high efficacy, but 50% of all children with a CH diagnosis were screening negative. The incidence of screening positive CH increased (1/3375 to 1/2222), and the incidence of screening negative CH decreased (1/2563 to 1/7841) after lowering the TSH screening threshold in 2009. Screening negative CH was associated with female sex, twinning, prematurity, low birth weight, birth defects, and need of neonatal intensive care, and 42% had transient disease. CONCLUSIONS: Despite high efficacy of the CH screening, 50% of children diagnosed as CH was screening negative. Although other factors influencing the incidence of the CH diagnosis cannot be ruled out, the incidence of screening negative CH decreased with lowering of the TSH threshold. Birth characteristics differed between screening positive and negative CH.

First Year of TREC-Based National SCID Screening in Sweden.

Screening for severe combined immunodeficiency (SCID) was introduced into the Swedish newborn screening program in August 2019 and here we report the results of the first year. T cell receptor excision circles (TRECs), kappa-deleting element excision circles (KRECs), and actin beta (ACTB) levels were quantitated by multiplex qPCR from dried blood spots (DBS) of 115,786 newborns and children up to two years of age, as an approximation of the number of recently formed T and B cells and sample quality, respectively. Based on low TREC levels, 73 children were referred for clinical assessment which led to the diagnosis of T cell lymphopenia in 21 children. Of these, three were diagnosed with SCID. The screening performance for SCID as the outcome was sensitivity 100%, specificity 99.94%, positive predictive value (PPV) 4.11%, and negative predictive value (NPV) 100%. For the outcome T cell lymphopenia, PPV was 28.77%, and specificity was 99.95%. Based on the first year of screening, the incidence of SCID in the Swedish population was estimated to be 1:38,500 newborns.

Integration of whole genome sequencing into a healthcare setting: high diagnostic rates across multiple clinical entities in 3219 rare disease patients.

BACKGROUND: We report the findings from 4437 individuals (3219 patients and 1218 relatives) who have been analyzed by whole genome sequencing (WGS) at the Genomic Medicine Center Karolinska-Rare Diseases (GMCK-RD) since mid-2015. GMCK-RD represents a long-term collaborative initiative between Karolinska University Hospital and Science for Life Laboratory to establish advanced, genomics-based diagnostics in the Stockholm healthcare setting. METHODS: Our analysis covers detection and interpretation of SNVs, INDELs, uniparental disomy, CNVs, balanced structural variants, and short tandem repeat expansions. Visualization of results for clinical interpretation is carried out in Scout-a custom-developed decision support system. Results from both singleton (84%) and trio/family (16%) analyses are reported. Variant interpretation is done by 15 expert teams at the hospital involving staff from three clinics. For patients with complex phenotypes, data is shared between the teams. RESULTS: Overall, 40% of the patients received a molecular diagnosis ranging from 19 to 54% for specific disease groups. There was heterogeneity regarding causative genes (n = 754) with some of the most common ones being COL2A1 (n = 12; skeletal dysplasia), SCN1A (n = 8; epilepsy), and TNFRSF13B (n = 4; inborn errors of immunity). Some causative variants were recurrent, including previously known founder mutations, some novel mutations, and recurrent de novo mutations. Overall, GMCK-RD has resulted in a large number of patients receiving specific molecular diagnoses. Furthermore, negative cases have been included in research studies that have resulted in the discovery of 17 published, novel disease-causing genes. To facilitate the discovery of new disease genes, GMCK-RD has joined international data sharing initiatives, including ClinVar, UDNI, Beacon, and MatchMaker Exchange. CONCLUSIONS: Clinical WGS at GMCK-RD has provided molecular diagnoses to over 1200 individuals with a broad range of rare diseases. Consolidation and spread of this clinical-academic partnership will enable large-scale national collaboration.

Clinical Presentation, Genetic Etiology, and Coenzyme Q10 Levels in 55 Children with Combined Enzyme Deficiencies of the Mitochondrial Respiratory Chain.

OBJECTIVES: To evaluate the clinical symptoms and biochemical findings and establish the genetic etiology in a cohort of pediatric patients with combined deficiencies of the mitochondrial respiratory chain complexes. STUDY DESIGN: Clinical and biochemical data were collected from 55 children. All patients were subjected to sequence analysis of the entire mitochondrial genome, except when the causative mutations had been identified based on the clinical picture. Whole exome sequencing/whole genome sequencing (WES/WGS) was performed in 32 patients. RESULTS: Onset of disease was generally early in life (median age, 6 weeks). The most common symptoms were muscle weakness, hypotonia, and developmental delay/intellectual disability. Nonneurologic symptoms were frequent. Disease causing mutations were found in 20 different nuclear genes, and 7 patients had mutations in mitochondrial DNA. Causative variants were found in 18 of the 32 patients subjected to WES/WGS. Interestingly, many patients had low levels of coenzyme Q10 in muscle, irrespective of genetic cause. CONCLUSIONS: Children with combined enzyme defects display a diversity of clinical symptoms with varying age of presentation. We established the genetic diagnosis in 35 of the 55 patients (64%). The high diagnostic yield was achieved by the introduction of massive parallel sequencing, which also revealed novel genes and enabled elucidation of new disease mechanisms.

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.

Newborn Screening for Presymptomatic Diagnosis of Complement and Phagocyte Deficiencies.

The clinical outcomes of primary immunodeficiencies (PIDs) are greatly improved by accurate diagnosis early in life. However, it is not common to consider PIDs before the manifestation of severe clinical symptoms. Including PIDs in the nation-wide newborn screening programs will potentially improve survival and provide better disease management and preventive care in PID patients. This calls for the detection of disease biomarkers in blood and the use of dried blood spot samples, which is a part of routine newborn screening programs worldwide. Here, we developed a newborn screening method based on multiplex protein profiling for parallel diagnosis of 22 innate immunodeficiencies affecting the complement system and respiratory burst function in phagocytosis. The proposed method uses a small fraction of eluted blood from dried blood spots and is applicable for population-scale performance. The diagnosis method is validated through a retrospective screening of immunodeficient patient samples. This diagnostic approach can pave the way for an earlier, more comprehensive and accurate diagnosis of complement and phagocytic disorders, which ultimately lead to a healthy and active life for the PID patients.

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.

MCEE Mutations in an Adult Patient with Parkinson's Disease, Dementia, Stroke and Elevated Levels of Methylmalonic Acid.

Methylmalonic aciduria (MMA-uria) is seen in several inborn errors of metabolism (IEM) affecting intracellular cobalamin pathways. Methylmalonyl-CoA epimerase (MCE) is an enzyme involved in the mitochondrial cobalamin-dependent pathway generating succinyl-CoA. Homozygous mutations in the corresponding MCEE gene have been shown in children to cause MCE deficiency with isolated MMA-uria and a variable clinical phenotype. We describe a 78-year-old man with Parkinson's disease, dementia and stroke in whom elevated serum levels of methylmalonic acid had been evident for many years. Metabolic work-up revealed intermittent MMA-uria and increased plasma levels of propionyl-carnitine not responsive to treatment with high-dose hydroxycobalamin. Whole genome sequencing was performed, with data analysis targeted towards genes known to cause IEM. Compound heterozygous mutations were identified in the MCEE gene, c.139C>T (p.Arg47X) and c.419delA (p.Lys140fs), of which the latter is novel. To our knowledge, this is the first report of an adult patient with MCEE mutations and MMA-uria, thus adding novel data to the possible phenotypical spectrum of MCE deficiency. Although clinical implications are uncertain, it can be speculated whether intermittent hyperammonemia during episodes of metabolic stress could have precipitated the patient's ongoing neurodegeneration attributed to Parkinson's disease.

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.

Surge of immune cell formation at birth differs by mode of delivery and infant characteristics-A population-based cohort study.

BACKGROUND: Birth by cesarean section is associated with increased risks of immune disorders. We tested whether establishment of immune function at birth relates to mode of delivery, taking other maternal and infant characteristics into account. METHODS AND FINDINGS: Using a prospectively collected database, we retrieved information on maternal and infant characteristics of 6,014 singleton infants delivered from February to April 2014 in Stockholm, Sweden, with gestational age ≥35 weeks, Apgar scores ≥7, and without congenital malformations or any neonatal morbidity. We linked our data to blood levels of T-cell receptor excision circles (TREC) and κ-deleting recombination excision circles (KREC), determined as part of a neonatal screening program for immune-deficiencies, and representing quantities of newly formed T- and B-lymphocytes. Multivariate logistic regression was used to calculate odds ratios (OR) with 95% confidence intervals (CI) for participants having TREC and KREC levels in the lowest quintile. Multivariate models were adjusted for postnatal age at blood sampling, and included perinatal (mode of delivery, infant sex, gestational age, and birth weight for gestational age), and maternal characteristics (age, parity, BMI, smoking, diabetes, and hypertensive disease). Low TREC was associated with cesarean section before labor (adjusted OR:1.32 [95% CI 1.08-1.62]), male infant sex (aOR:1.60 [1.41-1.83]), preterm birth at 35-36 weeks of gestation (aOR:1.89 [1.21-2.96]) and small for gestational age (aOR:1.67 [1.00-2.79]). Low KREC was associated with male sex (aOR:1.32 [1.15-1.50]), postterm birth at ≥42 weeks (aOR:1.43 [1.13-1.82]) and small for gestational age (aOR:2.89 [1.78-4.69]). Maternal characteristics showed no consistent associations with neonatal levels of either TREC or KREC. CONCLUSION: Cesarean section before labor was associated with lower T-lymphocyte formation, irrespective of maternal characteristics, pregnancy, and neonatal risk factors. The significance of a reduced birth-related surge in lymphocyte formation for future immune function and health remains to be investigated.

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.

[Hereditary metabolic diseases with onset in adulthood. Early and correct treatment of acute symptoms can be life-saving]. / Ärftliga metabola sjukdomar med symtomdebut i vuxen ålder - Tidig och korrekt behandling vid akuta symtom kan vara livräddande.

Inherited metabolic diseases usually present in the neonatal period or before school age. A growing portion of the disorders can be treated successfully, and an increasing number of patients are now treated in adult medicine. Several of the disorders also exist as attenuated variants without distinct symptoms in childhood. They can present as an acute onset event during metabolic stress in adulthood. We describe three patients with acute clinical decompensation in adulthood with severe sequelae and propose investigations to help diagnose such patients. One patient was unconscious from ammonia intoxication and developed severe neurological sequelae because of a defect in the urea cycle. A second patient had seizures caused by a defect in the conversion of homocysteine to methionine, resulting in lack of S-adenosylmethionine. A third patient had a stroke and grossly elevated homocysteine caused by undiagnosed homocystinuria. Recently there have been dramatic improvements in diagnostic methods, and new therapies are continuously being developed. Knowledge of these disorders is therefore of increasing importance also in adult medicine.

The ketogenic diet compensates for AGC1 deficiency and improves myelination.

The brain aspartate-glutamate carrier (AGC1) is specifically expressed in neurons, where it transports aspartate from the mitochondria to the cytosol, and plays a role in transfer of nicotinamide adenine dinucleotide (NADH)-reducing equivalents into the mitochondria as a part of the malate-aspartate shuttle. Deficient function of AGC1 underlies an inborn error of metabolism that presents with severe hypotonia, arrested psychomotor development, and seizures from a few months of age. In AGC1 deficiency, there is secondary hypomyelination due to lack of N-acetylaspartate (NAA), which is normally generated by acetylation of aspartate in the neuron and required for fatty acid synthesis by the adjacent oligodendrocyte. Based on experiences from AGC2 deficiency, we predicted that reduced glycolysis should compensate for the metabolic defect and allow resumed myelination in AGC1 deficiency. Carbohydrate restriction was therefore initiated in a patient with AGC1 deficiency at 6 years of age by introducing a ketogenic diet. The response was dramatic, clinically as well as radiologically. Psychomotor development showed clear improvement, and magnetic resonance imaging (MRI) indicated resumed myelination. This is the first successful treatment of secondary hypomyelination reported. Because AGC1 is driven by the proton gradient generated by the neuronal mitochondrial respiratory chain, the results have potential relevance for secondary hypomyelination in general.

Heat differentiated complement factor profiling.

Complement components and their cascade of reactions are important defense mechanisms within both innate and adaptive immunity. Many complement deficient patients still remain undiagnosed because of a lack of high throughput screening tools. Aiming towards neonatal proteome screening for immunodeficiencies, we used a multiplex profiling approach with antibody bead arrays to measure 9 complement proteins in serum and dried blood spots. Several complement components have been described as heat sensitive, thus their heat-dependent detectability was investigated. Using sera from 16 patients with complement deficiencies and 23 controls, we confirmed that the proteins C1q, C2, C3, C6, C9 and factor H were positively affected by heating, thus the identification of deficient patients was improved when preheating samples. Measurements of C7, C8 and factor I were negatively affected by heating and non-heated samples should be used in analysis of these components. In addition, a proof of concept study demonstrated the feasibility of labeling eluates from dried blood spots to perform a subsequent correct classification of C2-deficiencies. Our study demonstrates the potential of using multiplexed single binder assays for screening of complement components that open possibilities to expand such analysis to other forms of deficiencies.

Rescue of primary ubiquinone deficiency due to a novel COQ7 defect using 2,4-dihydroxybensoic acid.

BACKGROUND: Coenzyme Q is an essential mitochondrial electron carrier, redox cofactor and a potent antioxidant in the majority of cellular membranes. Coenzyme Q deficiency has been associated with a range of metabolic diseases, as well as with some drug treatments and ageing. METHODS: We used whole exome sequencing (WES) to investigate patients with inherited metabolic diseases and applied a novel ultra-pressure liquid chromatography-mass spectrometry approach to measure coenzyme Q in patient samples. RESULTS: We identified a homozygous missense mutation in the COQ7 gene in a patient with complex mitochondrial deficiency, resulting in severely reduced coenzyme Q levels We demonstrate that the coenzyme Q analogue 2,4-dihydroxybensoic acid (2,4DHB) was able to specifically bypass the COQ7 deficiency, increase cellular coenzyme Q levels and rescue the biochemical defect in patient fibroblasts. CONCLUSION: We report the first patient with primary coenzyme Q deficiency due to a homozygous COQ7 mutation and a potentially beneficial treatment using 2,4DHB.

Rapid pulsed whole genome sequencing for comprehensive acute diagnostics of inborn errors of metabolism.

BACKGROUND: Massively parallel DNA sequencing (MPS) has the potential to revolutionize diagnostics, in particular for monogenic disorders. Inborn errors of metabolism (IEM) constitute a large group of monogenic disorders with highly variable clinical presentation, often with acute, nonspecific initial symptoms. In many cases irreversible damage can be reduced by initiation of specific treatment, provided that a correct molecular diagnosis can be rapidly obtained. MPS thus has the potential to significantly improve both diagnostics and outcome for affected patients in this highly specialized area of medicine. RESULTS: We have developed a conceptually novel approach for acute MPS, by analysing pulsed whole genome sequence data in real time, using automated analysis combined with data reduction and parallelization. We applied this novel methodology to an in-house developed customized work flow enabling clinical-grade analysis of all IEM with a known genetic basis, represented by a database containing 474 disease genes which is continuously updated. As proof-of-concept, two patients were retrospectively analysed in whom diagnostics had previously been performed by conventional methods. The correct disease-causing mutations were identified and presented to the clinical team after 15 and 18 hours from start of sequencing, respectively. With this information available, correct treatment would have been possible significantly sooner, likely improving outcome. CONCLUSIONS: We have adapted MPS to fit into the dynamic, multidisciplinary work-flow of acute metabolic medicine. As the extent of irreversible damage in patients with IEM often correlates with timing and accuracy of management in early, critical disease stages, our novel methodology is predicted to improve patient outcome. All procedures have been designed such that they can be implemented in any technical setting and to any genetic disease area. The strategy conforms to international guidelines for clinical MPS, as only validated disease genes are investigated and as clinical specialists take responsibility for translation of results. As follow-up in patients without any known IEM, filters can be lifted and the full genome investigated, after genetic counselling and informed consent.

Epidemiology of lysosomal storage diseases in Sweden.

AIM: There are more than 50 inherited lysosomal storage diseases (LSDs), and this study examined the incidence of clinically diagnosed LSDs in Sweden. METHODS: The number of patients diagnosed during 1980-2009 was compiled from the registries of the two Swedish diagnostic laboratories that cover the whole country. RESULTS: We identified 433 patients during the 30-year period, with a total incidence of one in every 6100 births and identified fairly constant annual diagnoses during the last 20 years. Krabbe disease was the most common (one in 39 000) followed by Gaucher disease (one in 47 000), metachromatic leukodystrophy and Salla disease. Gaucher disease was more frequent in Sweden than other European countries, due to a founder effect of the mutation (p.L444P) in northern Sweden. Metachromatic leukodystrophy was one of the most common LSDs, in common with other countries. Salla disease, which is very rare elsewhere, was the fourth most common, stemming from a founder mutation in the Salla region of northern Finland brought to Sweden by immigration. CONCLUSION: The collective incidence of LSDs in Sweden was essentially equal to other European countries, but with a somewhat different disease pattern. Our findings have implications for diagnostic algorithms and treatment strategies.