Higher precision, first tier newborn screening for metachromatic leukodystrophy using 16:1-OH-sulfatide.

Newborn screening (NBS) for metachromatic leukodystrophy (MLD) is based on first-tier measurement of sulfatides in dried blood spots (DBS) followed by second-tier measurement of arylsulfatase A in the same DBS. This approach is very precise with 0-1 false positives per ∼30,000 newborns tested. Recent data reported here shows that the sulfatide molecular species with an α-hydroxyl, 16­carbon, mono-unsaturated fatty acyl group (16:1-OH-sulfatide) is superior to the original biomarker 16:0-sulfatide in reducing the number of first-tier false positives. This result is consistent across 4 MLD NBS centers. By measuring 16:1-OH-sulfatide alone or together with 16:0-sulfatide, the estimated false positive rate is 0.048% and is reduced essentially to zero with second-tier arylsulfatase A activity assay. The false negative rate is predicted to be extremely low based on the demonstration that 40 out of 40 newborn DBS from clinically-confirmed MLD patients are detected with these methods. The work shows that NBS for MLD is extremely precise and ready for deployment. Furthermore, it can be multiplexed with several other inborn errors of metabolism already tested in NBS centers worldwide.

Long-Term Evaluation of Biomarkers in the Czech Cohort of Gaucher Patients.

A personalized treatment decision for Gaucher disease (GD) patients should be based on relevant markers that are specific to GD, play a direct role in GD pathophysiology, exhibit low genetic variation, reflect the therapy, and can be used for all patients. Thirty-four GD patients treated with enzyme replacement therapy (ERT) or substrate reduction therapy (SRT) were analyzed for platelet count, chitotriosidase, and tartrate-resistant acid phosphatase activity in plasma samples, and quantitative measurement of Lyso-Gb1 was performed in dried blood spots. In our ERT and SRT study cohorts, plasma lyso-GL1 correlated significantly with chito-triosidase (ERT: r = 0.55, p < 0.001; SRT: r = 0.83, p < 0.001) and TRAP (ERT: r = 0.34, p < 0.001; SRT: r = 0.88, p < 0.001), irrespective of treatment method. A platelet count increase was associated with a Lyso-Gb1 decrease in both treatment groups (ERT: p = 0.021; SRT: p = 0.028). The association of Lyso-Gb1 with evaluated markers was stronger in the SRT cohort. Our results indicate that ERT and SRT in combination or in a switch manner could offer the potential of individual drug effectiveness for particular GD symptoms. Combination of the key biomarker of GD, Lyso-Gb1, with other biomarkers can offer improved response assessment to long-term therapy.

Comparison of sequential versus concurrent chemoradiation regimens in non-metastatic muscle-invasive bladder cancer.

PURPOSE: The treatment approach for non-metastatic bladder cancer is guided by an invasion of the muscular layer of the bladder wall. Radical cystectomy is the recommended treatment for muscle-invasive disease. However, it has considerable morbidity and mortality and is not suited for many patients. Trimodality therapy consisting of chemoradiation after transurethral resection of bladder tumor offers a definitive approach with bladder-sparing potential. However, there is a lack of research defining the optimal combination of chemotherapy and radiation in this setting. MATERIALS AND METHODS: We extracted patient data from the National Cancer Database to compare survival outcomes and demographic factors in 2,227 non-metastatic bladder cancer patients who were treated with chemotherapy sequential to or concurrently with radiation. Sequential treatment was defined as chemotherapy beginning >14 days before radiation, and concurrent was defined as beginning within 14 days of the first radiation. RESULTS: The sequential treatment group patients were younger (mean age, 74 vs. 78 years; p < 0.001) with more advanced disease. We found no difference in overall survival between patients who received chemotherapy sequential to radiation and those who received concurrent chemoradiation only (p = 0.533). CONCLUSION: Our data are concordant with a previous prospective study, and support that chemotherapy prior to radiation does not decrease survival outcomes relative to patients receiving only concurrent chemoradiation. Given that the sequential group had an overall higher stage but no difference in survival, downstaging chemotherapy prior to radiation may be helpful in these patients. Further studies including a larger, multi-institutional clinical trial are indicated to support clinical decision-making.

Vanadium oxide and a sharp onset of cold-trapping on a giant exoplanet.

The abundance of refractory elements in giant planets can provide key insights into their formation histories1. Owing to the low temperatures of the Solar System giants, refractory elements condense below the cloud deck, limiting sensing capabilities to only highly volatile elements2. Recently, ultra-hot giant exoplanets have allowed for some refractory elements to be measured, showing abundances broadly consistent with the solar nebula with titanium probably condensed out of the photosphere3,4. Here we report precise abundance constraints of 14 major refractory elements on the ultra-hot giant planet WASP-76b that show distinct deviations from proto-solar and a sharp onset in condensation temperature. In particular, we find nickel to be enriched, a possible sign of the accretion of the core of a differentiated object during the evolution of the planet. Elements with condensation temperatures below 1,550 K otherwise closely match those of the Sun5 before sharply transitioning to being strongly depleted above 1,550 K, which is well explained by nightside cold-trapping. We further unambiguously detect vanadium oxide on WASP-76b, a molecule long suggested to drive atmospheric thermal inversions6, and also observe a global east-west asymmetry7 in its absorption signals. Overall, our findings indicate that giant planets have a mostly stellar-like refractory elemental content and suggest that temperature sequences of hot Jupiter spectra can show abrupt transitions wherein a mineral species is either present or completely absent if a cold trap exists below its condensation temperature8.

Importance to include differential diagnostics for acid sphingomyelinase deficiency (ASMD) in patients suspected to have to Gaucher disease.

The clinical manifestation of sphingolipidosis leads often to misclassification between acid sphingomyelinase deficiency (ASMD) and Gaucher disease. In this multicenter, prospective study, we investigated a cohort of 31,838 individuals suspected to have Gaucher disease, due to clinical presentation, from 61 countries between 2017 and 2022. For all samples, both Acid-ß-glucocerebrosidase and acid sphingomyelinase enzyme activities were measured in dried blood spot specimens by tandem mass spectrometry followed by genetic confirmatory testing in potential positive cases. In total, 5933 symptomatic cases showed decreased enzyme activities and were submitted for genetic confirmatory testing. 1411/5933 (24%) cases were finally identified with Gaucher disease and 550/5933 (9%) with ASMD. Most of the confirmed ASMD cases were newborns and children below 2 years of age (63%). This study reveals that one in four cases suspected for Gaucher disease is diagnosed with ASMD. An early appropriate diagnostic work-up is essential because of the availability of a recently approved enzyme replacement therapy for ASMD. In conclusion, a diagnostic strategy using differential biochemical testing including genetic confirmatory testing in clinically suspected cases for sphingolipidosis is highly recommended.

Nationwide screening of Fabry disease in patients with hypertrophic cardiomyopathy in Czech Republic.

AIMS: Fabry disease (FD) is a rare X-linked genetic disorder caused by α-galactosidase A (AGALA) deficiency. Whereas 'classic' variant has multisystemic manifestation, the more recently described 'later-onset' variant is characterized by predominant cardiac involvement that often mimics hypertrophic cardiomyopathy (HCM). METHODS AND RESULTS: Consecutive unrelated patients with HCM were screened for FD in 16 (out of 17) cardiac centres in the Czech Republic covering specialized cardiology care from June 2017 to December 2018. AGALA activity and globotriaosylsphingosine (lyso-Gb3 ) levels were measured in all subjects using the dry blood spot method. FD was suspected in male patients with AGALA activity <1.2 µmol/h/L and in females with either low AGALA activity or lyso-Gb3  > 3.5 ng/mL. Positive screening results were confirmed by genetic testing. We evaluated 589 patients (390 males, 66%) with HCM (mean maximal myocardial thickness 19.1 ± 4.3 mm). The average age was 58.4 ± 14.7 years. In total, 17 patients (11 males, 6 females) had a positive screening result, and subsequently, six of them (four males and two females) had a genetically confirmed pathogenic GLA mutation (total prevalence of 1.02%). Five of these patients were carrying the p.N215S mutation known to cause a typical later-onset cardiac FD. CONCLUSIONS: We confirmed the prevalence of FD repeatedly reported in previous screening programmes (approximately 1% irrespective of gender) in a non-selected HCM population in Central Europe. Our findings advocate a routine screening for FD in all adult patients with HCM phenotype including both genders. The dry blood spot method used led to identification of clearly pathogenic variants.

Lyso-Gb3 associates with adverse long-term outcome in patients with Fabry disease.

BACKGROUND: Fabry disease (FD) is a rare X-linked lysosomal storage disease caused by mutations in the α-galactosidase A gene (GLA) leading to deficiency of α-galactosidase A and ultimately in progressive glycosphingolipid accumulation, especially globotriaosylceramide (Gb3) and its deacylated derivative globotriaosylsphingosine (Lyso-Gb3). The aim of the study was to assess plasma Lyso-Gb3 levels as a possible factor associated with adverse outcomes in FD. METHODS: In a cohort of 66 patients with genetically confirmed FD (26 males and 40 females), we analysed serum Lyso-Gb3 as a factor associated with adverse clinical outcomes in a long-term study. The main outcome was a composite endpoint of incident kidney replacement therapy, atrial fibrillation, pacemaker and/or implantable cardioverter defibrillator, cerebrovascular events or death, whichever occurred first. RESULTS: During the median follow-up time of 68 (40-80) months, events occurred in 19 (29%) of the patients. In a Cox multivariate regression analysis, Lyso-Gb3 levels (HR 4.62 (1.55 to 13.81); p=0.006) and the pretreatment exposure to Lyso-Gb3 (HR 3.41 (1.11 to 10.49); p=0.03) (both per SD increase) were significantly associated with adverse outcomes. If pretreatment Lyso-Gb3 exposure was added to multivariable logistic regression models containing age, sex, phenotype and enzyme replacement therapy as other covariates with the composite outcome as dependent variable, the area under the curve for the composite outcome significantly improved from 0.72 to 0.86 (p comparison=0.04). CONCLUSION: Lyso-Gb3 is a significant risk factor associated with important clinical events. Whether treatment-related amelioration of Lyso-Gb3 levels will be associated with improved long-term outcome needs to be established in prospective intervention trials.

Molecular basis of mucopolysaccharidosis IVA (Morquio A syndrome): A review and classification of GALNS gene variants and reporting of 68 novel variants.

Mucopolysaccharidosis IVA (MPS IVA, Morquio A syndrome) is a rare autosomal recessive lysosomal storage disorder caused by mutations in the N-acetylgalactosamine-6-sulfatase (GALNS) gene. We collected, analyzed, and uniformly summarized all published GALNS gene variants, thus updating the previous mutation review (published in 2014). In addition, new variants were communicated by seven reference laboratories in Europe, the Middle East, Latin America, Asia, and the United States. All data were analyzed to determine common alleles, geographic distribution, level of homozygosity, and genotype-phenotype correlation. Moreover, variants were classified according to their pathogenicity as suggested by ACMG. Including those previously published, we assembled 446 unique variants, among which 68 were novel, from 1190 subjects (including newborn screening positive subjects). Variants' distribution was missense (65.0%), followed by nonsense (8.1%), splicing (7.2%), small frameshift deletions(del)/insertions(ins) (7.0%), intronic (4.0%), and large del/ins and complex rearrangements (3.8%). Half (50.4%) of the subjects were homozygous, 37.1% were compound heterozygous, and 10.7% had only one variant detected. The novel variants underwent in silico analysis to evaluate their pathogenicity. All variants were submitted to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) to make them publicly available. Mutation updates are essential for the correct molecular diagnoses, genetic counseling, prenatal and preimplantation diagnosis, and disease management.

At-Risk Testing for Pompe Disease Using Dried Blood Spots: Lessons Learned for Newborn Screening.

Pompe disease (GSD II) is an autosomal recessive disorder caused by deficiency of the lysosomal enzyme acid-α-glucosidase (GAA, EC 3.2.1.20), leading to generalized accumulation of lysosomal glycogen especially in the heart, skeletal, and smooth muscle, and the nervous system. It is generally classified based on the age of onset as infantile (IOPD) presenting during the first year of life, and late onset (LOPD) when it presents afterwards. In our study, a cohort of 13,627 samples were tested between January 2017 and December 2018 for acid-α-glucosidase (GAA, EC 3.2.1.20) deficiency either by fluorometry or tandem mass spectrometry (MS). Testing was performed for patients who displayed conditions of unknown etiology, e.g., CK elevations or cardiomyopathy, in the case of infantile patients. On average 8% of samples showed activity below the reference range and were further assessed by another enzyme activity measurement or molecular genetic analysis. Pre-analytical conditions, like proper drying, greatly affect enzyme activity, and should be assessed with measurement of reference enzyme(s). In conclusion, at-risk testing can provide a good first step for the future introduction of newborn screening for Pompe disease. It yields immediate benefits for the patients regarding the availability and timeliness of the diagnosis. In addition, the laboratory can introduce the required methodology and gain insights in the evaluation of results in a lower throughput environment. Finally, awareness of such a rare condition is increased tremendously among local physicians which can aid in the introduction newborn screening.

Linear basal cell nevus with a novel mosaic PTCH1 mutation.

The patched tumor suppressor gene (PTCH1) encodes a receptor, which is a key component of the hedgehog signalling pathway. Mutations in PTCH1 are implicated in the development of sporadic basal cell carcinomas (BCC), as well as those in Gorlin Syndrome. Rarely, BCCs may develop in a linear pattern along lines of Blaschko due to cutaneous mosaicism. In cases in which there are other features of Gorlin syndrome, genomic analysis has demonstrated lesional mutations in the Hedgehog signalling pathway. Causative mutations, however, have not been firmly demonstrated in the cases of linear and segmental BCCs in otherwise healthy individuals. Herein, we report a case of a 31 year-old Caucasian woman with linear development of multiple superficial BCCs in a Blaschkoid distribution without other characteristic findings of Gorlin syndrome. Genomic analysis of lesional skin by whole-exome sequencing identified a novel heterozygous mutation PTCH1: NM_000264.3, Exon 15, c.2336-2337insGGTAGGA, p.Asp779Glufs*13 in PTCH1, shared by two discrete samples within the lesion, while no mutations were found in the non-lesional skin or peripheral blood. Given the young age of our patient and linear distribution of BCCs on non-sun exposed skin, our findings suggest segmental mosaicism. The patient was treated with topical 5% imiquimod with histologically confirmed clearance of BCCs in 2 months.

α-Mannosidosis - An underdiagnosed lysosomal storage disease in individuals with an 'MPS-like' phenotype.

Individuals affected by alpha-Mannosidosis suffer from similar clinical symptoms such as respiratory infections, skeletal changes as patients with mucopolysaccharidoses (MPS). α-Mannosidosis is considered as an ultra-rare disorders and also diagnostic testing is often limited. With the availability of novel therapies and easy-to-access diagnostic tests (e.g. Tandem mass spectrometry) using dried blood spots for both enzymatic and genetic testing, the chance for the development of a better understanding of disease and awareness may be triggered. In a pilot study, we have investigated 1010 residual dried blood spot samples from individuals suspicious to MPS. In these study cohort, 158/1010 individuals were genetically confirmed for MPS. Additional biochemical and genetic confirmatory testing for α-mannosidases revealed four individuals with a final diagnosis of α-mannosidosis. This unexpected high number of individuals with α-mannosidosis demonstrated the urgent need of taking this rare disorder in clinical and diagnostic consideration particularly in patients suspicious to MPS.

Investigating the suitability of high-resolution mass spectrometry for newborn screening: identification of hemoglobinopathies and ß-thalassemias in dried blood spots.

A fast and reliable method for the determination of hemoglobinopathies and thalassemias by high-resolution accurate mass spectrometry (HRAM/MS) is presented. The established method was verified in a prospective clinical study (HRAM/MS vs. high-pressure liquid chromatography [HPLC]) of 5335 de-identified newborn samples from the Hamburg area. The analytical method is based on a dual strategy using intact protein ratios for thalassemias and tryptic digest fragments for the diagnosis of hemoglobinopathies. Due to the minimal sample preparation and the use of flow injection, the assay can be considered as a high-throughput screening approach for newborn screening programs (2 min/sample). Using a simple dried blood spot (DBS) extraction (tryptic digest buffer), the following results were obtained: (1) a carrier incidence of 1:100 newborns (35 FAS, nine FAC, eight FAD and two FAE), and (2) no homozygous affected patient was detected. Using the HRAM/MS protocol, an unknown Hb mutation was identified and confirmed by genetic testing. In addition to greater specificity toward rare mutations and ß-thalassemia, the low price/sample (1-2€) as well as an automated data processing represent the major benefits of the described HRAM/MS method.

A click-flipped enzyme substrate boosts the performance of the diagnostic screening for Hunter syndrome.

We report on the unexpected finding that click modification of iduronyl azides results in a conformational flip of the pyranose ring, which led to the development of a new strategy for the design of superior enzyme substrates for the diagnostic assaying of iduronate-2-sulfatase (I2S), a lysosomal enzyme related to Hunter syndrome. Synthetic substrates are essential in testing newborns for metabolic disorders to enable early initiation of therapy. Our click-flipped iduronyl triazole showed a remarkably better performance with I2S than commonly used O-iduronates. We found that both O- and triazole-linked substrates are accepted by the enzyme, irrespective of their different conformations, but only the O-linked product inhibits the activity of I2S. Thus, in the long reaction times required for clinical assays, the triazole substrate substantially outperforms the O-iduronate. Applying our click-flipped substrate to assay I2S in dried blood spots sampled from affected patients and random newborns significantly increased the confidence in discriminating between these groups, clearly indicating the potential of the click-flip strategy to control the biomolecular function of carbohydrates.

Diagnostic strategy for females suspected of Fabry disease.

A total of 11 948 females suspicious of Fabry disease were tested by a combined biochemical and genetic approach. The enzyme activity, together with the concentration of lyso-GL-3 (lyso-Gb3) biomarker in dried blood spots (DBS), substantially improved the diagnostic detection of Fabry disease in females compared to the enzyme activity alone. Abnormal values for both were highly suspicious of Fabry disease (97% positive predictive value [PPV], similar to PPV in males). In cases with one abnormal biochemical value, elevated lyso-GL-3 is a far more important indicator than low enzyme activity (39% PPV vs 6% PPV). Cases with clearly negative results for both biochemical parameters are unlikely to have Fabry disease, even in clinically highly suspicious cases.

Negative screening of Fabry disease in patients with conduction disorders requiring a pacemaker.

Identification of Fabry disease (FD) in cardiac patients has been restricted so far to patients with left ventricular hypertrophy. Conduction problems are frequent in FD and could precede other manifestations, offering a possible earlier diagnosis.We studied the prevalence of FD in 188 patients < 70 years with conduction problems requiring pacemaker implantation. Although classical manifestations of FD were not rare, no patient with FD was identified. Screening efforts should not be conducted in this population.

Vaginal Ureaplasma parvum serovars and spontaneous preterm birth.

BACKGROUND: Ureaplasma species (spp) are the bacteria most often isolated from the amniotic cavity of women with preterm labor or preterm premature rupture of membranes; thus, the link between intrauterine Ureaplasma spp infection and adverse pregnancy outcome clearly is established. However, because vaginal Ureaplasma spp colonization is very common in pregnant women, the reason that these microorganisms cause ascending infections in some cases but remain asymptomatic in most pregnancies is not clear. Previous studies suggested an association between vaginal colonization with Ureaplasma parvum as opposed to U urealyticum and preterm delivery. However, because of the high frequency of vaginal Ureaplasma spp colonization during pregnancy, additional risk factors are needed to select a group of women who might benefit from treatment. OBJECTIVE: To further identify pregnant women who are at increased risk for preterm delivery, the aim of the present study was to investigate U parvum serovar-specific pathogenicity in a large clinical cohort. STUDY DESIGN: We serotyped 1316 samples that were positive for U parvum using a high-resolution melt polymerase chain reaction assay, and results were correlated with pregnancy outcome. RESULTS: Within U parvum positive samples, serovar 3 was the most common isolate (43.3%), followed by serovar 6 (31.4%) and serovar 1 (25.2%). There was a significantly increased risk for spontaneous preterm birth at very low (<32 weeks gestation; P<.005) and extremely low (<28 weeks gestation; P<.005) gestational age in the group with vaginal U parvum serovar 3 colonization compared with the control group of pregnant women who tested negative for vaginal Ureaplasma spp colonization. This association was found for neither serovar 1 nor serovar 6. The combination of vaginal U parvum serovar 3 colonization and diagnosis of bacterial vaginosis in early pregnancy or a history of preterm birth further increased the risk for adverse pregnancy outcome. CONCLUSION: Colonization with U parvum serovar 3, but not serovar 1 or serovar 6, in early pregnancy is associated with preterm delivery at very and extremely low gestational age. The combination of U parvum serovar 3 colonization and a history of preterm birth or bacterial vaginosis further increases the risk for spontaneous preterm birth at low gestational age and may define a target group for therapeutic intervention studies.

Asparagine levels in the cerebrospinal fluid of children with acute lymphoblastic leukemia treated with pegylated-asparaginase in the induction phase of the AIEOP-BFM ALL 2009 study.

Asparagine levels in cerebrospinal fluid and serum asparaginase activity were monitored in children with acute lymphoblastic leukemia treated with pegylated-asparaginase. The drug was given intravenously at a dose of 2,500 IU/m2 on days 12 and 26. Serum and cerebrospinal fluid samples obtained on days 33 and 45 were analyzed centrally. Since physiological levels of asparagine in the cerebrospinal fluid of children and adolescents are 4-10 µmol/L, in this study asparagine depletion was considered complete when the concentration of asparagine was ≤0.2 µmol/L, i.e. below the lower limit of quantification of the assay used. Over 24 months 736 patients (AIEOP n=245, BFM n=491) and 903 cerebrospinal fluid samples (n=686 on day 33 and n=217 on day 45) were available for analysis. Data were analyzed separately for the AIEOP and BFM cohorts and yielded superimposable results. Independently of serum asparaginase activity levels, cerebrospinal fluid asparagine levels were significantly reduced during the investigated study phase but only 28% of analyzed samples showed complete asparagine depletion while relevant levels, ≥1 µmol/L, were still detectable in around 23% of them. Complete cerebrospinal fluid asparagine depletion was found in around 5-6% and 33-37% of samples at serum asparaginase activity levels <100 and ≥ 1,500 IU/L, respectively. In this study cerebrospinal fluid asparagine levels were reduced during pegylated-asparaginase treatment, but complete depletion was only observed in a minority of patients. No clear threshold of serum pegylated-asparaginase activity level resulting in complete cerebrospinal fluid asparagine depletion was identified. The consistency of the results found in the two independent data sets strengthen the observations of this study. Details of the treatment are available in the European Clinical Trials Database at https://www.clin-icaltrialsregister.eu/ctr-search/trial/2007-004270-43/IT.

Low Prevalence Estimates of Late-Onset Glycogen Storage Disease Type II in French-Speaking Belgium are not Due to Missed Diagnoses.

BACKGROUND: Late-onset glycogen storage disease type II is associated with variable muscle phenotypes. Epidemiological data suggest that its prevalence is lower in Belgium than in bordering countries like The Netherlands. OBJECTIVE: We investigated whether such low estimated prevalence is due to missed diagnoses. METHODS: We screened 100 patients with muscle phenotypes of undetermined origin using a dried blood spot test for alpha-acid glucosidase (GAA) activity. Patients with low activity at screening were re-tested by the same method and, if low activity was confirmed, GAA gene analysis was performed. RESULTS: The screening test revealed lower than normal GAA activity in 15 patients, but in only two of them it was low enough to be considered in the disease range. Retesting confirmed lower than normal GAA activity in five patients, but in all of them it was above the disease range. A single patient carried a heterozygous known pathogenic GAA mutation, whose significance in this case remains undetermined. CONCLUSIONS: We conclude that reported low prevalence estimates in Belgium are not likely to be due to an underdiagnosis bias. Lower prevalence compared to neighbouring The Netherlands may be due to different ethnic stratification of our patients. Diagnostic strategies should keep into account the expected prevalence of a disease in specific populations.

Pulmonary involvement in Fabry disease: effect of plasma globotriaosylsphingosine and time to initiation of enzyme replacement therapy.

INTRODUCTION: Anderson-Fabry disease (AFD) is an X-linked lysosomal storage disorder caused by mutations of GLA gene leading to reduced α-galactosidase activity and resulting in a progressive accumulation of globotriaosylceramide (Gb3) and its deacylated derivative, globotriaosyl-sphingosine (Lyso-Gb3). Plasma Lyso-Gb3 levels serve as a disease severity and treatment monitoring marker during enzyme replacement therapy (ERT). METHODS: Adult patients with AFD who had yearly pulmonary function tests between 1999 and 2015 were eligible for this observational study. Primary outcome measures were the change in z-score of forced expiratory volume in the first second (FEV1) and FEV1/FVC over time. Plasma Lyso-Gb3 levels and the age of ERT initiation were investigated for their association with lung function decline. RESULTS: Fifty-three patients (42% male, median (range) age at diagnosis of AFD 34 (6-61) years in men, 34 (13-67) in women) were included. The greatest decrease of FEV1/FVC z-scores was observed in Classic men (-0.048 per year, 95% CI -0.081 to -0.014), compared with the Later-Onset men (+0.013,95% CI -0.055 to 0.082), Classic women (-0.008, 95% CI -0.035 to +0.020) and Later-Onset women (-0.013, 95% CI -0.084 to +0.058). Cigarette smoking (P=0.022) and late ERT initiation (P=0.041) were independently associated with faster FEV1 decline. FEV1/FVC z-score decrease was significantly reduced after initiation of ERT initiation (-0.045 compared with -0.015, P=0.014). Furthermore, there was a trend towards a relevant influence of Lyso-Gb3 (P=0.098) on airflow limitation with age. CONCLUSION: Early ERT initiation seems to preserve pulmonary function. Plasma Lyso-Gb3 is maybe a useful predictor for airflow limitation. Classic men need a closer monitoring of the lung function.