cDNA sequencing increases the molecular diagnostic yield in Chediak-Higashi syndrome

Abstract

Introduction: Chediak-Higashi syndrome (CHS) is rare autosomal recessive disorder caused by bi-allelic variants in the Lysosomal Trafficking Regulator (LYST) gene. Diagnosis is established by the detection of pathogenic variants in LYST in combination with clinical evidence of disease. Conventional molecular genetic testing of LYST by genomic DNA (gDNA) Sanger sequencing detects the majority of pathogenic variants, but some remain undetected for several individuals clinically diagnosed with CHS. In this study, cDNA Sanger sequencing was pursued as a complementary method to identify variant alleles that are undetected by gDNA Sanger sequencing and to increase molecular diagnostic yield. Methods: Six unrelated individuals with CHS were clinically evaluated and included in this study. gDNA Sanger sequencing and cDNA Sanger sequencing were performed to identify pathogenic LYST variants. Results: Ten novel LYST alleles were identified, including eight nonsense or frameshift variants and two in-frame deletions. Six of these were identified by conventional gDNA Sanger sequencing; cDNA Sanger sequencing was required to identify the remaining variant alleles. Conclusion: By utilizing cDNA sequencing as a complementary technique to identify LYST variants, a complete molecular diagnosis was obtained for all six CHS patients. In this small CHS cohort, the molecular diagnostic yield was increased, and canonical splice site variants identified from gDNA Sanger sequencing were validated by cDNA sequencing. The identification of novel LYST alleles will aid in diagnosing patients and these molecular diagnoses will also lead to genetic counseling, access to services and treatments and clinical trials in the future.

Keywords: LYST; molecular diagnosis; monogenic diseases; oculocutaneous albinism; personalized medicine; rare disorders; splicing abnormalities.

FIGURE 1

Clinical images of individuals with Chediak-Higashi syndrome (CHS) and cellular phenotype and LYST gene expression in primary cells from individuals with CHS (A) Partial oculocutaneous albinism often manifests as silvery hair and atypical pigment clumping of the hair shaft observed on light microscopy in individuals with CHS. (B) Peripheral blood smear demonstrating pathognomonic giant inclusions in polymorphonuclear leukocytes in CHD22, CHD25, and CHD36 and a single giant inclusion in a lymphocyte in CHD25 (arrowhead) (C) Primary fibroblasts (upper panel) and melanocytes (lower panel) were stained with LAMP3/CD63 antibodies to visualize lysosomal membranes. Primary fibroblasts were stained with phalloidin to visualize cell boundaries marked by F-actin. Lysosomes in unaffected control fibroblasts are distributed throughout the cell, while fibroblasts from individuals with CHS show enlarged lysosomes restricted to the perinuclear area. In melanocytes from CHD25, enlarged lysosomes were distributed throughout the cell compared to unaffected control melanocytes. Scale bar = 20 microns (D) Relative LYST mRNA expression in primary fibroblasts (upper panel) or melanocytes (lower panel). For the fibroblast expression data, the data are presented as a box and whisker plot relative to the highest LYST-expressing unaffected control with all individual mean data points represented. Group relative LYST expression differences were tested using a two-tailed Mann-Whitney test. For the melanocyte expression data, the data are presented as the mean of three technical replicates relative to the highest LYST-expressing unaffected control and error bars represent one standard deviation. Expression of HPRT1, POLR2A, and TBP were used as internal controls to normalize gene expression. Abbreviations: *, p < 0.05.

FIGURE 2

LYST variant alleles in individuals with Chediak-Higashi syndrome (CHS) identified by gDNA and cDNA Sanger sequencing (A) Schematics of the LYST mRNA (NM_000081.3) and LYST protein (NP_000072.2) denoting the relative locations of the LYST variants identified by gDNA and cDNA Sanger sequencing. Previously reported variants (white), novel variants identified by gDNA Sanger sequencing (black), and novel variants identified by cDNA Sanger sequencing (red) are represented in the schematic and include frameshift (square),nonsense (diamond), missense (circle), and in-frame (triangle) variants. Functional domains of LYST include the WD repeats (light grey), the PH domain (medium grey), and the BEACH domain (dark grey), and the corresponding regions are color-coded in the LYST mRNA schematic. The LYST mRNA schematic is not drawn to scale, while the LYST protein schematic is drawn to scale (B) Chromatograms generated through cDNA Sanger sequencing of LYST, including the c.9785_9925del (exon 43 deletion) variant allele identified in CHD3, the c.11039_11195del (exon 51 deletion) variant allele identified in CHD5, the c.8359_8389del (partial exon 32 deletion) variant allele identified in CHD22, the homozygous c.9163_9560del (exon 39 and 40 deletion) variant allele identified in CHD25, the c.8152_8358del (exon 31 deletion) variant allele identified in CHD34, and the c.9926_10029del (partial exon 44 deletion) variant allele identified in CHD36. RT-PCR products were cloned to obtain sequence data for the c.11039_11195del variant allele. Abbreviations: BEACH domain, beige and Chediak-Higashi syndrome domain; LYST, lysosomal trafficking regulator; PH domain, pleckstrin homology domain.