Chediak-Higashi syndrome with early developmental delay resulting from paternal heterodisomy of chromosome 1

Abstract

Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disease characterized by variable oculocutaneous albinism, immunodeficiency, mild bleeding diathesis, and an accelerated lymphoproliferative state. Abnormal lysosome-related organelle membrane function leads to the accumulation of large intracellular vesicles in several cell types, including granulocytes, melanocytes, and platelets. This report describes a severe case of CHS resulting from paternal heterodisomy of chromosome 1, causing homozygosity for the most distal nonsense mutation (p.E3668X, exon 50) reported to date in the LYST/CHS1 gene. The mutation is located in the WD40 region of the CHS1 protein. The patient's fibroblasts expressed no detectable CHS1. Besides manifesting the classical CHS findings, the patient exhibited hypotonia and global developmental delays, raising concerns about other effects of heterodisomy. An interstitial 747 kb duplication on 6q14.2-6q14.3 was identified in the propositus and paternal samples by comparative genomic hybridization. SNP genotyping revealed no additional whole chromosome or segmental isodisomic regions or other dosage variations near the crossover breakpoints on chromosome 1. Unmasking of a separate autosomal recessive cause of developmental delay, or an additive effect of the paternal heterodisomy, could underlie the severity of the phenotype in this patient.

FIG. 1. Clinical findings of patient CHS-16

(A) Iris transillumination (caused by light that is transmitted back through the iris when it is shone through the pupil) in CHS-16, indicating a lack of pigment; (B) Hypopigmentation of the retina in CHS-16; (C) Microscopic examination of the hair revealed the presence of small, homogeneously distributed pigment clumps (arrows); (D-F) Wright stains of peripheral smear showing an eosinophil, polymorphonuclear leukocyte and lymphocyte with giant intracytoplasmic granules, as well as Dohle bodies (E) (100×). (G) Whole mount electron microscopy of platelets from the patient’s blood showing absent dense bodies (10,000×). (H) Only two normal-sized dense bodies (arrows) were present in one of the normal-sized platelets (13,000×).

FIG. 2. Mutation analysis and protein expression

(A) Sequencing of the CHS1 gene revealed an apparently homozygous G>T nucleotide substitution in exon 50 (arrow) in patient CHS-16, resulting in the replacement of a glutamic acid (GAA), with a stop codon (TAA), p.E3668X mutation. This change was found in the heterozygous state in the paternal, but not the maternal sample; (B) Western blot of protein extracts derived from CHS-16, control and CHS-4 fibroblasts labeled with anti-rabbit anti-CHS1 and anti-mouse anti-alpha-Tubulin Ab (loading control, 55 kDa). CHS-16 fibroblasts do not express CHS1 protein (~430 kDa, lane 1).

FIG. 3. Haplotype analysis using chromosome 1 polymorphic markers show primary heterodisomy of chromosome 1

A total of 29 polymorphic markers on chromosome 1 were employed for haplotype analysis. Small areas of heterozygosity were observed at the 1p telomere and around the centromere, where markers from both the paternal homologues were detected (solid black bars: paternal allele 1; solid gray bars: paternal allele 2; dashed/striped bars: maternal alleles; solid lines combining the bars represent areas where the markers were non-informative, i.e. the father was homozygous for the respective marker. Asterisk represents a single marker that does not appear to derive from the father, and may indicate variability due to slippage during replication.

FIG. 4. CHS-16 fibroblasts have enlarged lysosomes

(A) LAMP-3 staining (green) in control fibroblasts shows lysosomes distributed throughout the cell (arrows). (B) Enlarged view of LAMP-3 in control fibroblasts. (C) LAMP-3 (green) in CHS-16 fibroblasts shows accumulation of enlarged lysosomes in the perinuclear area (arrows). (D) Enlarged view of LAMP-3 in CHS-16 fibroblasts, showing some normal sized lysosomes in the peripheral area (arrowheads). Co-staining with BODIPY 558/568 phalloidin (red) visualizes the cell boundaries; the DAPI stain (blue) visualizes the nucleus. Scale bars 20 μm.