Selective screening for lysosomal storage disorders in a large cohort of minorities of African descent shows high prevalence rates and novel variants

Population studies point to regional and ethnicity-specific differences in genetic predisposition for some lysosomal storage disorders (LSDs). The aim of the study was to determine the prevalence of the three treatable forms of lysosomal storage disorders (Gaucher disease [GD], Pompe disease [PD], and Fabry disease [FD]) in a cohort of mostly urban-dwelling individuals of African ancestry, a previously unknown genetic landscape for LSDs. Large-scale selective multistep biochemical and genetic screening was performed in patients seeking healthcare for various health concerns. Fluorimetric enzyme assays for GD, PD, and FD were performed on dried blood spots. Targeted gene sequencing was performed on samples that showed significantly lower enzyme activities (\u3c10% of control mean) after two tiers of enzymatic screening. A total of 5287 unique samples representing a cross section of patients who visited Howard University Hospital and College of Medicine from 2015 to 2017 were included in the study. Study samples were obtained from a population where ~90% reported as African-American, ~5% Hispanic, and \u3c5% Caucasian or other. Regarding GD, three subjects had either homozygous or heterozygous mutations in the GBA gene. As to PD, eight subjects were either homozygous or compound heterozygous for GAA mutations, including three novel mutations: (a) c.472 A \u3e G; p.T158A, (b) c.503G \u3e T; p.R168L, (c) c.1985del. Regarding FD, two subjects had pathogenic GLA mutations, and four had single nucleotide polymorphisms in the 5\u27UTR, previously implicated in modulating gene expression. The findings highlight a higher incidence of abnormal enzyme levels and pathogenic mutations in the target population reflecting ancestry-based specific genotype and phenotype variations

Effect of early or delayed therapy on immune irregularities.

<p>GD patients were sub-divided into those who received early vs. delayed intervention after their diagnosis and plotted for specific immune phenotypes. B cell transitional cells expressed as CD21^Dim (A), IgA producing B cells (B), NKT cells (C) and dendritic cell (D) percentages in GD patients with delayed or early intervention are plotted.</p

Time of Initiating Enzyme Replacement Therapy Affects Immune Abnormalities and Disease Severity in Patients with Gaucher Disease

<div><p>Gaucher disease (GD) patients often present with abnormalities in immune response that may be the result of alterations in cellular and/or humoral immunity. However, how the treatment and clinical features of patients impact the perturbation of their immunological status remains unclear. To address this, we assessed the immune profile of 26 GD patients who were part of an enzyme replacement therapy (ERT) study. Patients were evaluated clinically for onset of GD symptoms, duration of therapy and validated outcome measures for ERT. According to DS3 disease severity scoring system criteria, they were assigned to have mild, moderate or severe GD. Flow cytometry based immunophenotyping was performed to analyze subsets of T, B, NK, NKT and dendritic cells. GD patients showed multiple types of immune abnormalities associated to T and B lymphocytes with respect to their subpopulations as well as memory and activation markers. Skewing of CD4 and CD8 T cell numbers resulting in lower CD4/CD8 ratio and an increase in overall T cell activation were observed. A decrease in the overall B cells and an increase in NK and NKT cells were noted in the GD patients compared to controls. These immune alterations do not correlate with GD clinical type or level of biomarkers. However, subjects with persistent immune alterations, especially in B cells and DCs correlate with longer delay in initiation of ERT (ΔTX). Thus, while ERT may reverse some of these immune abnormalities, the immune cell alterations become persistent if therapy is further delayed. These findings have important implications in understanding the immune disruptions before and after treatment of GD patients.</p></div

Correlation between immune alterations and ΔTX, DS3 and combined scores.

<p>GD patients were sub-categorized based on whether or not they show persistent B-cell (A), T-cell (B), NKT cells (CD16/CD56+ T cells) (C) and dendritic cell (D) immune alterations and plotted against the delay in initiation of ERT (ΔTX), disease severity (DS3) and a combined score (ΔTX+DS3).</p

Activation markers in Th and Tc cells.

<p>CD4+ T (A) and CD8+ T cells (B) were further analyzed for expression of activation markers (CCR4, CXCR3), chemokine receptor (CCR6) and chemoattractant (CRTH2) which play a role in T-cell mediated inflammation.</p

Correlation between delay in treatment initiation and disease severity scores.

<p>Delay in initiation of treatment for GD (ΔTX) was plotted against disease severity score (DS3) for each patient. The trend line shows a positive Pearson correlation coefficient between the two values (r value = 0.55, P = 0.0018) (A). Subjects were divided based on their DS3 scores as being mild/moderate (DS3<5) or marked (DS3>6) and their ΔTX values were plotted. Unpaired Student’s t-test revealed P value = 0.0036 (**) indicating very significant difference (B).</p

Inflammatory cytokine expression on T cells.

<p>Memory T cells were further analyzed for co-expression of inflammatory chemokine receptors (CCR4, CCR5 and CCR6) which play a role in T cell mediated inflammation.</p

Clinical evaluations and DS3 scoring.

<p>Clinical evaluations and DS3 scoring.</p

T-lymphocytes and subsets in GD patients.

<p>T-lymphocytes (CD3+) from peripheral blood of GD patients and normal controls were assessed using flow cytometry and plotted as fraction of total lymphocytes (A). T helper cells (CD3+/CD4+) (B) and cytotoxic T cells (CD3+/CD8+) (C) were calculated and a ratio of CD4 to CD8 cells was plotted (D). Similarly memory subsets of CD4 (CD3+/CD4+/CD45RO+) and CD8 T cells (CD3+/CD8+/CD45RO+) were calculated and plotted against normal controls (E, F). Unpaired student’s t-test was performed to calculate significance values and included in the plots where significant difference between GD and normal controls was observed.</p