Inherited Metabolic Disorders: Efficacy of Enzyme Assays on Dried Blood Spots for the Diagnosis of Lysosomal Storage Disorders

Abstract

High consanguinity rates, poor access to accurate diagnostic tests, and costly therapies are the main causes of increased burden of lysosomal storage disorders (LSDs) in developing countries. Therefore, there is a major unmet need for accurate and economical diagnostic tests to facilitate diagnosis and consideration of therapies before irreversible complications occur. In cross-country study, we utilized dried blood spots (DBS) of 1,033 patients clinically suspected to harbor LSDs for enzymatic diagnosis using modified fluorometric assays from March 2013 through May 2015. Results were validated by demonstrating reproducibility, testing in different sample types (leukocytes/plasma/skin fibroblast), mutation study, or measuring specific biomarkers. Thirty percent (307/1,033) were confirmed to have one of the LSDs tested. Reference intervals established unambiguously identified affected patients. Correlation of DBS results with other biological samples (n = 172) and mutation studies (n = 74) demonstrated 100% concordance in Gaucher, Fabry, Tay Sachs, Sandhoff, Niemann-Pick, GM1, Neuronal ceroid lipofuscinosis (NCL), Fucosidosis, Mannosidosis, Mucopolysaccharidosis (MPS) II, IIIb, IVa, VI, VII, and I-Cell diseases, and 91.4% and 88% concordance in Pompe and MPS-I, respectively. Gaucher and Pompe are the most common LSDs in India and Pakistan, followed by MPS-I in both India and Sri Lanka. Study demonstrates utility of DBS for reliable diagnosis of LSDs. Diagnostic accuracy (97.6%) confirms veracity of enzyme assays. Adoption of DBS will overcome significant hurdles in blood sample transportation from remote regions. DBS enzymatic and molecular diagnosis should become the standard of care for LSDs to make timely diagnosis, develop personalized treatment/monitoring plan, and facilitate genetic counseling.

Presentation and treatments for Mucopolysaccharidosis Type II (MPS II; Hunter Syndrome)

“…Establishment of affordable and efficient newborn screenings will likely lead to early diagnosis and early treatment [18, 74, 159]. …”

“…Wang’s group used a novel approach with the addition of a synthetic substrate for the enzyme to a buffer rehydrated with 2 disks from a DBS. The enzyme-generated product was then measured by tandem mass spectrometry (MS/MS) [68,74]. To exclude multiple sulfatase deficiency, other lysosomal enzymes should be measured [75,76].…”

Mucopolysaccharidosis IIIB (Sanfilippo syndrome B) in a commercial emu (Dromaius novaehollandiae) flock

“…While blood samples from the breeders and tissues from necropsied birds were used as DNA sources for screening emus for the NAGLU mutation in this study, immature feathers containing pulp or dried blood spots might be simpler to collect and yet serve as well. Dried blood spots have been used for economical diagnostic screening of people in developing countries to identify a variety of lysosomal diseases including MPS IIIB (Verma et al , 2017). Cheek swabs or cytobrushes and dried blood spot samples are used routinely for genetic screening of dogs, cats, and people and could work in emus.…”

False Positive Diagnosis of Lysosomal Storage Disease Based on Dried Blood Spot Sample; Leucocyte Number of a Challenging Factor

“…The strict control of storage conditions (such as humidity, temperature and so on), the correct preparation of the sample, sample blank correction and participation in quality control programs are recommended by many authors in order to validate these methods. Recently, the specificity and sensitivity of DBS enzyme assays to diagnose LSD have been reported as 95.2% (92-96%) and 85% (64-100%) respectively (5,6). Even though all above mentioned factors were controlled, the false positive rates are still high because of individual factors, such as pseudo deficiency due to isozymes and so on.…”