Sanfilippo syndrome type B: Analysis of patients diagnosed by the MPS Brazil Network.

Mucopolysaccharidosis type IIIB is a rare autosomal recessive disorder characterized by deficiency of the enzyme N-acetyl-alpha-d-glucosaminidase (NAGLU), caused by biallelic pathogenic variants in the NAGLU gene, which leads to storage of heparan sulfate and a series of clinical consequences which hallmark is neurodegeneration. In this study clinical, epidemiological, and biochemical data were obtained from MPS IIIB patients diagnosed from 2004-2019 by the MPS Brazil Network ("Rede MPS Brasil"), which was created with the goal to provide an easily accessible and comprehensive investigation of all MPS types. One hundred and ten MPS IIIB patients were diagnosed during this period. Mean age at diagnosis was 10.9 years. Patients were from all over Brazil, with a few from abroad, with a possible cluster of MPS IIIB identified in Ecuador. All patients had increased urinary levels of glycosaminoglycans and low NAGLU activity in blood. Main clinical symptoms reported at diagnosis were coarse facies and neurocognitive regression. The most common variant was p.Leu496Pro (30% of alleles). MPS IIIB seems to be relatively frequent in Brazil, but patients are diagnosed later than in other countries, and reasons for that probably include the limited awareness about the disease by health professionals and the difficulties to access diagnostic tests, factors that the MPS Brazil Network is trying to mitigate.

Genotype-phenotype studies in a large cohort of Brazilian patients with Hunter syndrome.

Mucopolysaccharidosis type II (MPS II) is an X-linked inherited disease caused by pathogenic variants in the IDS gene, leading to deficiency of the lysosomal enzyme iduronate-2-sulfatase and consequent widespread storage of glycosaminoglycans, leading to several clinical consequences, with progressive manifestations which most times includes cognitive decline. MPS II has wide allelic and clinical heterogeneity and a complex genotype-phenotype correlation. We evaluated data from 501 Brazilian patients diagnosed with MPS II from 1982 to 2020. We genotyped 280 of these patients (55.9%), which were assigned to 206 different families. Point mutations were present in 70% of our patients, being missense variants the most frequent. We correlated the IDS pathogenic variants identified with the phenotype (neuronophatic or non-neuronopathic). Except for two half-brothers, there was no discordance in the genotype-phenotype correlation among family members, nor among MPS II patients from different families with the same single base-pair substitution variant. Mothers were carriers in 82.0% of the cases. This comprehensive study of the molecular profile of the MPS II cases in Brazil sheds light on the genotype-phenotype correlation and helps the better understanding of the disease and the prediction of its clinical course, enabling the provision of a more refined genetic counseling to the affected families.

COVID-19 impact on the diagnosis of Inborn Errors of Metabolism: Data from a reference center in Brazil.

The COVID-19 pandemic led to the reorganization of health care in several countries, including Brazil. Inborn Errors of Metabolism (IEM) are a group of rare and difficult to diagnose genetic diseases caused by pathogenic variants in genes that code for enzymes, cofactors, or structural proteins affecting different metabolic pathways. The aim of this study was to evaluate how COVID-19 affected the diagnosis of patients with IEM during the first year of the pandemic in Brazil comparing two distinct periods: from March 1st, 2019 to February 29th, 2020 (TIME A) and from March 1st, 2020 to February 28th, 2021 (TIME B), by the analysis of the number of tests and diagnoses performed in a Reference Center in South of Brazil. In the comparison TIME A with TIME B, we observe a reduction in the total number of tests performed (46%) and in the number of diagnoses (34%). In both periods analyzed, mucopolysaccharidoses (all subtypes combined) was the most frequent LD suspected and/or confirmed. Our data indicates a large reduction in the number of tests requested for the investigation of IEM and consequently a large reduction in the number of diagnoses caused by the COVID-19 pandemic leading to a significant underdiagnosis of IEM.

Updated birth prevalence and relative frequency of mucopolysaccharidoses across Brazilian regions.

The mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by 11 enzyme deficiencies, classified into seven types. Data on the birth prevalence of each MPS type are available for only a few countries, and the totality of cases may be underestimated. To determine the epidemiological profile of MPS in each Brazilian region, we analyzed data collected between 1982 and 2019 by a national reference laboratory and identified 1,652 patients. Using data between 1994 and 2018, the birth prevalence (by 100,000 live births) for MPS was 1.57. MPS II was the most common type of MPS in Brazil, and its birth prevalence was 0.48 (0.94 considering only male births). Regarding the number of cases per region, MPS II was the most frequent in the North and Center-West (followed by MPS VI), and also in the Southeast (followed by MPS I); MPS I and MPS II were the most common types in the South; and MPS VI was the most common in the Northeast (followed by MPS II). The differences observed in the relative frequencies of MPS types across Brazilian regions are likely linked to founder effect, endogamy, and consanguinity, but other factors may be present and need further investigation.

Multiplex testing for the screening of lysosomal storage disease in urine: Sulfatides and glycosaminoglycan profiles in 40 cases of sulfatiduria.

PURPOSE: To describe an efficient and effective multiplex screening strategy for sulfatide degradation disorders and mucolipidosis type II/III (MLII/III) using 3 mL of urine. METHODS: Glycosaminoglycans were analyzed by liquid chromatography-tandem mass spectrometry. Matrix assisted laser desorption/ionization-time of flight tandem mass spectrometry was used to identify free oligosaccharides and identify 22 ceramide trihexosides and 23 sulfatides, which are integrated by 670 calculated ratios. Collaborative Laboratory Integrated Reports (CLIR; https://clir.mayo.edu) was used for post-analytical interpretation of the complex metabolite profile and to aid in the differential diagnosis of abnormal results. RESULTS: Multiplex analysis was performed on 25 sulfatiduria case samples and compiled with retrospective data from an additional 15 cases revealing unique patterns of biomarkers for each disorder of sulfatide degradation (MLD, MSD, and Saposin B deficiency) and for MLII/III, thus allowing the formulation of a novel algorithm for the biochemical diagnosis of these disorders. CONCLUSIONS: Comprehensive and integrated urine screening could be very effective in the initial workup of patients suspected of having a lysosomal disorder as it covers disorders of sulfatide degradation and narrows down the differential diagnosis in patients with elevated glycosaminoglycans.

Newborn screening for lysosomal disorders in Brazil: A pilot study using customized fluorimetric assays.

Lysosomal storage disorders (LSDs) are a group of genetic disorders characterized by deficiency of specific lysosomal enzymes. In general, patients are clinically normal at birth, and progressively develop severe signs and symptoms. Diagnosis is usually made several years after onset of manifestations, preventing patients to have the benefits of the early treatment. Newborn screening programs are being considered for LSDs to allow early diagnosis and treatment. The present study evaluated the feasibility of a customized screening approach based on modified fluorometric assays with reduced amounts of reagents, substrates and samples for: mucopolysaccharidosis (MPS) type I (MPS I), MPS VI, Fabry, Gaucher, and Pompe diseases. We also evaluated the advantages of including blood chitotriosidase and urinary glycosaminoglycans in the protocol. By the measurement of the specific disease-associated enzymes (plus blood chitotriosidase and urinary glycosaminoglycans) we analyzed 834 de-identified DBS of unselected newborns. No positive case was detected, and the false-positive rates were low. Taking into consideration the limitations of this methodology, we believe that, after defining proper cutoffs, it could be a viable alternative to provide NBS for LSDs by laboratories that may not be able to afford the commercial methods available.

Lysosomal diseases: Overview on current diagnosis and treatment.

Lysosomal diseases (LDs), also known as lysosomal storage diseases (LSDs), are a heterogeneous group of conditions caused by defects in lysosomal function. LDs may result from deficiency of lysosomal hydrolases, membrane-associated transporters or other non-enzymatic proteins. Interest in the LD field is growing each year, as more conditions are, or will soon be treatable. In this article, we review the diagnosis of LDs, from clinical suspicion and screening tests to the identification of enzyme or protein deficiencies and molecular genetic diagnosis. We also cover the treatment approaches that are currently available or in development, including hematopoietic stem cell transplantation, enzyme replacement therapy, small molecules, and gene therapy.

Information and Diagnosis Networks - tools to improve diagnosis and treatment for patients with rare genetic diseases.

Brazil is a country of continental dimensions and most genetic services are concentrated in the Southeast and South, including the Medical Genetics Service of the Hospital de Clínicas de Porto Alegre (MGS/HCPA). As many areas on the country do not have adequate medical genetics support, networks were designed to extend the service of the MGS/HCPA reference center. This paper presents the information and diagnosis networks that have their headquarters at MGS/HCPA: SIAT (National Information System on Teratogenic Agents), SIEM (Information Service on Inborn Errors of Metabolism), Alô Genética (Hello Genetics - Medical Genetics Information Service for Primary Health Care Professionals); Rede MPS Brasil (MPS-Mucopolysaccharidosis Brazil Network); Rede EIM Brasil (IEM-Inborn Errors of Metabolism Brazil Network), Rede NPC Brasil (Niemann-Pick C - NPC Brazil Network), Rede DLD Brasil (LSD-Lysosomal Storage Disorders Brazil Network), Rede DXB (MSUD-Maple Syrup Urine Disease Network), RedeBRIM (Brazilian Network of Reference and Information in Microdeletion Syndromes Project), Rede Neurogenética (Neurogenetics Network), and Rede Brasileira de Câncer Hereditário (Brazilian Hereditary Cancer Network). These tools are very useful to provide access to a qualified information and/or diagnostic service for specialized and non-specialized health services, bypassing difficulties that preclude patients to access reference centers.

Population medical genetics: translating science to the community.

Rare genetic disorders are currently in the spotlight due to the elevated number of different conditions and significant total number of affected patients. The study of these disorders is extremely helpful for the elucidation of physiological processes related with complex disorders. Isolated populations are instrumental for the study of genetic disorders, considering their homogeneity and high proportion of affected patients in a small geographic area. These favorable conditions lead to the creation of a new discipline, known as "population medical genetics", which integrates medical genetics, population genetics, epidemiological genetics and community genetics. In order to develop practical activities in this new discipline, the National Institute of Population Medical Genetics (INaGeMP) was created in 2008 in Brazil. INaGeMP has developed several tools and funded numerous research activities. In this review, we highlight three successful projects developed in the first 10 years of INaGeMP activities (2008-2018): a newborn screening pilot study for MPS VI in Northeast Brazil, the study of Machado-Joseph disease in Brazilian families with Azorian ancestry, and the high twinning rate in a small town in southern Brazil. The results of these projects in terms of scientific output and contributions to the affected communities highlight the success and importance of INaGeMP.

Oxidative and nitrative stress and pro-inflammatory cytokines in Mucopolysaccharidosis type II patients: effect of long-term enzyme replacement therapy and relation with glycosaminoglycan accumulation.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease caused by a deficient activity of iduronate-2-sulfatase, leading to abnormal accumulation of glycosaminoglycans (GAG). The main treatment for MPS II is enzyme replacement therapy (ERT). Previous studies described potential benefits of six months of ERT against oxidative stress in patients. Thus, the aim of this study was to investigate oxidative, nitrative and inflammatory biomarkers in MPS II patients submitted to long term ERT. It were analyzed urine and blood samples from patients on ERT (mean time: 5.2years) and healthy controls. Patients presented increased levels of lipid peroxidation, assessed by urinary 15-F2t-isoprostane and plasmatic thiobarbituric acid-reactive substances. Concerning to protein damage, urinary di-tyrosine (di-Tyr) was increased in patients; however, sulfhydryl and carbonyl groups in plasma were not altered. It were also verified increased levels of urinary nitrate+nitrite and plasmatic nitric oxide (NO) in MPS II patients. Pro-inflammatory cytokines IL-1ß and TNF-α were increased in treated patients. GAG levels were correlated to di-Tyr and nitrate+nitrite. Furthermore, IL-1ß was positively correlated with TNF-α and NO. Contrastingly, we did not observed alterations in erythrocyte superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities, in reduced glutathione content and in the plasmatic antioxidant capacity. Although some parameters were still altered in MPS II patients, these results may suggest a protective role of long-term ERT against oxidative stress, especially upon oxidative damage to protein and enzymatic and non-enzymatic defenses. Moreover, the redox imbalance observed in treated patients seems to be GAG- and pro-inflammatory cytokine-related.

Elevation of glycosaminoglycans in the amniotic fluid of a fetus with mucopolysaccharidosis VII.

OBJECTIVE: The aim of this study was to quantify glycosaminoglycans (GAGs) in amniotic fluid (AF) from an MPS VII fetus compared with age-matched fetuses obtained from normal pregnancies. METHOD: Disaccharides were measured by liquid chromatography tandem mass spectrometry, compared to age-matched controls. Enzyme assay was performed in AF supernatant or cultured amniocytes. GUSB was analyzed by next generation sequencing using Ion Torrent Personal Genome Machine with a customized panel. RESULTS: No activity of ß-glucuronidase was detected in fetal cells. The pregnancy was spontaneously terminated in the third trimester. Genetic studies identified a homozygous mutation of p.N379D (c.1135A > G) in the GUSB gene. Liquid chromatography tandem mass spectrometry showed that chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate levels were markedly increased in the MPS VII AF, compared to those in age-matched control AF (dermatan sulfate, heparan sulfate, and chondroitin-6-sulfate more than 10 × than age-matched controls; chondroitin-4-sulfate and keratan sulfate more than 3 times higher). CONCLUSION: This is the first report of specific GAG analysis in AF from an MPS VII fetus, indicating that GAG elevation in AF occurs by 21 weeks of gestation and could be an additional tool for prenatal diagnosis of MPS VII and potentially other MPS types. © 2017 John Wiley & Sons, Ltd.

Relative frequency and estimated minimal frequency of Lysosomal Storage Diseases in Brazil: Report from a Reference Laboratory.

Lysosomal storage diseases (LSDs) comprise a heterogeneous group of more than 50 genetic conditions of inborn errors of metabolism (IEM) caused by a defect in lysosomal function. Although there are screening tests for some of these conditions, diagnosis usually depends on specific enzyme assays, which are only available in a few laboratories around the world. A pioneer facility for the diagnosis of IEM and LSDs was established in the South of Brazil in 1982 and has served as a reference service since then. Over the past 34 years, samples from 72,797 patients were referred for investigation of IEM, and 3,211 were confirmed as having an LSD (4.41%, or 1 in 22), with 3,099 of these patients originating from Brazil. The rate of diagnosis has increased over time, in part due to the creation of diagnostic networks involving a large number of Brazilian services. These cases, referred from Brazilian regions, provide insight about the relative frequency of LSDs in the country. The large amount of data available allows for the estimation of the minimal frequency of specific LSDs in Brazil. The reported data could help to plan health care policies, as there are specific therapies available for most of the cases diagnosed.

Oxidative stress and inflammation in mucopolysaccharidosis type IVA patients treated with enzyme replacement therapy.

Mucopolysaccharidosis type IVA (MPS IVA) is an inborn error of glycosaminoglycan (GAG) catabolism due to the deficient activity of N-acetylgalactosamine-6-sulfate sulfatase that leads to accumulation of the keratan sulfate and chondroitin 6-sulfate in body fluids and in lysosomes. The pathophysiology of this lysosomal storage disorder is not completely understood. The aim of this study was to investigate oxidative stress parameters, pro-inflammatory cytokine and GAG levels in MPS IVA patients. We analyzed urine and blood samples from patients under ERT (n=17) and healthy age-matched controls (n=10-15). Patients presented a reduction of antioxidant defense levels, assessed by a decrease in glutathione content and by an increase in superoxide dismutase activity in erythrocytes. Concerning lipid and protein damage, it was verified increased urine isoprostanes and di-tyrosine levels and decreased plasma sulfhydryl groups in MPS IVA patients compared to controls. MPS IVA patients showed higher DNA damage than control group and this damage had an oxidative origin in both pyrimidine and purine bases. Interleukin 6 was increased in patients and presented an inverse correlation with GSH levels, showing a possible link between inflammation and oxidative stress in MPS IVA disease. The data presented suggest that pro-inflammatory and pro-oxidant states occur in MPS IVA patients even under ERT. Taking these results into account, supplementation of antioxidants in combination with ERT can be a tentative therapeutic approach with the purpose of improving the patient's quality of life. To the best of our knowledge, this is the first study relating MPS IVA patients with oxidative stress.

Non-immune hydrops fetalis: A prospective study of 53 cases.

Non-immune hydrops fetalis (NIHF) is a symptom caused by a heterogeneous group of conditions. Diagnostic investigations may constitute a real challenge. This study aimed to evaluate prospectively and systematically a series of NIHF cases using a research protocol expanded for studying inborn errors of metabolism (IEM) during 2 years-2010 and 2011. We also reviewed the frequency of IEM among the NIHF reported in literature. A clinical or etiopathogenic diagnosis was reached in 46 (86.8%) of the 53 studied cases. The main diagnostic groups were chromosomal anomalies (28.3%), syndromic (18.9%), isolated cardiovascular anomaly (7.5%) and congenital infection (7.5%). Metabolic causes were found in 5.7%, all lysosomal storage disorders (LSD). In seven (13.2%), no diagnosis was found in part because of incomplete evaluation. The hydrops was identified prenatally in 90.5% of cases. In 5.7% a spontaneous and complete resolution of the hydrops occurred during pregnancy. Overall mortality was 75.5%. The IEM frequency in the present study (5.7%) was higher than that usually reported. We suggest performing studies directed to IEMs if the more common causes are excluded.

Diagnosing mucopolysaccharidosis IVA.

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is an autosomal recessive lysosomal storage disorder resulting from a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) activity. Diagnosis can be challenging and requires agreement of clinical, radiographic, and laboratory findings. A group of biochemical genetics laboratory directors and clinicians involved in the diagnosis of MPS IVA, convened by BioMarin Pharmaceutical Inc., met to develop recommendations for diagnosis. The following conclusions were reached. Due to the wide variation and subtleties of radiographic findings, imaging of multiple body regions is recommended. Urinary glycosaminoglycan analysis is particularly problematic for MPS IVA and it is strongly recommended to proceed to enzyme activity testing even if urine appears normal when there is clinical suspicion of MPS IVA. Enzyme activity testing of GALNS is essential in diagnosing MPS IVA. Additional analyses to confirm sample integrity and rule out MPS IVB, multiple sulfatase deficiency, and mucolipidoses types II/III are critical as part of enzyme activity testing. Leukocytes or cultured dermal fibroblasts are strongly recommended for enzyme activity testing to confirm screening results. Molecular testing may also be used to confirm the diagnosis in many patients. However, two known or probable causative mutations may not be identified in all cases of MPS IVA. A diagnostic testing algorithm is presented which attempts to streamline this complex testing process.

Intraperitoneal implant of recombinant encapsulated cells overexpressing alpha-L-iduronidase partially corrects visceral pathology in mucopolysaccharidosis type I mice.

BACKGROUND AIMS: Mucopolysaccharidosis type I (MPS I) is characterized by deficiency of the enzyme alpha-L-iduronidase (IDUA) and storage of glycosaminoglycans (GAG) in several tissues. Current available treatments present limitations, thus the search for new therapies. Encapsulation of recombinant cells within polymeric structures combines gene and cell therapy and is a promising approach for treating MPS I. METHODS: We produced alginate microcapsules containing baby hamster kidney (BHK) cells overexpressing IDUA and implanted these capsules in the peritoneum of MPS I mice. RESULTS: An increase in serum and tissue IDUA activity was observed at early time-points, as well as a reduction in GAG storage; however, correction in the long term was only partially achieved, with a drop in the IDUA activity being observed a few weeks after the implant. Analysis of the capsules obtained from the peritoneum revealed inflammation and a pericapsular fibrotic process, which could be responsible for the reduction in IDUA levels observed in the long term. In addition, treated mice developed antibodies against the enzyme. CONCLUSIONS: The results suggest that the encapsulation process is effective in the short term but improvements must be achieved in order to reduce the immune response and reach a stable correction.

Recombinant encapsulated cells overexpressing alpha-L-iduronidase correct enzyme deficiency in human mucopolysaccharidosis type I cells.

Mucopolysaccharidosis I (MPS I) is an autosomal recessive lysosomal storage disease due to deficient α-L-iduronidase (IDUA) activity. It results in the accumulation of the glycosaminoglycans (GAGs) heparan and dermatan sulfate and leads to several clinical manifestations. Available treatments are limited in their efficacy to treat some aspects of the disease. Thus, new approaches have been studied for the treatment of MPS I. Here, we tested the ability of recombinant baby hamster kidney cells transfected with human IDUA cDNA in correcting skin fibroblasts from MPS I patients in vitro. Our results showed an increase in IDUA activity in MPS I fibroblasts after 15, 30 and 45 days of coculture with the capsules. Cytological analysis showed a marked reduction in GAG storage within MPS I cells. Enzyme uptake by the fibroblasts was blocked in a dose-dependent manner with mannose-6-phosphate (M6P), indicating that cells use the M6P receptor to internalize the recombinant enzyme. Capsules were effective in correcting MPS I cells even after a 12-month period of cryopreservation. Taken together, our results indicate that cell encapsulation is a potential approach for treatment of MPS I. This approach becomes particularly interesting as a complementary approach, since the capsules could be implanted in sites which current treatments available are not able to reach. Future studies will focus on the efficacy of this approach in vivo.

A patient presenting a 22q13 deletion associated with an apparently balanced translocation t(16;22): An illustrative case in the investigation of patients with low ARSA activity.

A 10-year-old speechless, mentally deficient male, with low arylsulfatase A (ARSA) activity, and presumably, methachromatic leukodystrophy, underwent genetic evaluation. As the clinical picture was not compatible with this diagnosisan ARSA gene and chromosome analysis were performed, showing the presence of a pseudodeficiency ARSA allele and a de novo apparently balanced t(16;22)(p11.2;q13) translocation. A deletion on the long arm of chromosome 22 encompassing the ARSA gene, as shown by FISH and array-CGH, indicated a 22q13 deletion syndrome. This case illustrates the importance of detailed cytogenetic investigation in patients presenting low arylsulfatase A activity and atypical/unspecific clinical features.

Experience of the NPC Brazil Network with a Comprehensive Program for the Screening and Diagnosis of Niemann-Pick Disease Type C.

Niemann-Pick disease type C (NPC) is a lysosomal disorder caused by impaired cholesterol metabolism. Levels of lysosphingomyelin 509 (LysoSM509) have been shown elevated in dried blood spots (DBS) of NPC and acid sphingomyelinase deficiency patients. In this study, we report our experience using a two-tier approach (1st tier is the quantification of lysoSM509 by ultra-performance liquid chromatography tandem mass spectrometry followed by the 2nd tier with next-generation sequencing of the NPC1 and NPC2 genes). DBS samples from 450 suspected patients were received by the NPC Brazil network. Of these, 33 samples had elevated levels of lysoSM509, and in 25 of them, variants classified as pathogenic, likely pathogenic, or of unknown significance were identified in the NPC1 or NPC2 genes by next-generation sequencing. The quantification of lysoSM509 in DBS as a first-tier test for the diagnosis of NPC followed by molecular analysis of the NPC1 and NPC2 genes almost doubled the detection rate when compared to the performance of chitotriosidase activity as a first-tier biomarker, and it could likely be increased with the addition of a third tier with MLPA of the two genes involved. This strategy seems suitable for the neonatal screening (NBS) of NPC if this disease is eventually adopted by NBS programs.

Measurement of sulfatides in the amniotic fluid supernatant: A useful tool in the prenatal diagnosis of metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal disorder caused by deficiency of arylsulfatase A (ARSA), leading to an accumulation of sulfatides. Sulfatides have been quantified in urine, dried blood spots (DBS), and tissues of patients with MLD. Newborn screening (NBS) for MLD has already been proposed based on a two-tier approach with the quantification of sulfatides in DBS followed by the quantification of ARSA by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Prenatal screening for MLD is also crucial, and sulfatide quantification in amniotic fluid (AF) can aid diagnosis. The prenatal study was initiated due to a family history of MLD at 19 weeks of gestation. ARSA was quantified in cultured amniocytes. C16:0 sulfatide was quantified by LC-MS/MS in the supernatant of AF. Molecular analysis of the ARSA gene was performed in cultured amniocytes. ARSA was deficient in fetal cells, and C16:0 sulfatides were significantly elevated in comparison to age-matched controls (3-fold higher). Genetic studies identified the c.465+1G>A variant in homozygosis in the ARSA gene. Our study shows that sulfatides can be quantified in the supernatant of AF of MLD fetuses, and it could potentially aid in a faster and more accurate diagnosis of MLD patients.