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

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.

Diagnosis of Chediak Higashi disease in a 67-year old woman.

Chediak-Higashi disease is a rare disease caused by bi-allelic mutations in the lysosomal trafficking regulator gene, LYST. Individuals typically present in early childhood with partial oculocutaneous albinism, a bleeding diathesis, recurrent infections secondary to immune dysfunction, and risk of developing hemophagocytic lymphohistiocytosis (HLH). Without intervention, mortality is high in the first decade of life. However, some individuals with milder phenotypes have attenuated hematologic and immunologic presentations, and lower risk of HLH. Both classic and milder phenotypes develop progressive neurodegeneration in early adulthood. Here we present a remarkable patient diagnosed with Chediak-Higashi disease at age 67, many decades after the diagnosis is usually established. Diagnosis was suspected by observing the pathognomonic granules within leukocytes, and confirmed by identification of bi-allelic mutations in LYST, reduced LYST mRNA expression, enlarged lysosomes within fibroblasts, and decreased NK cell lytic activity. This case further expands the phenotype of Chediak-Higashi disease and highlights the need for increased awareness. Individuals with milder phenotypes may escape early diagnosis, but identification is important for close monitoring of potential complications, and to further our understanding of the function of LYST.

An Ultraconserved Brain-Specific Enhancer Within ADGRL3 (LPHN3) Underpins Attention-Deficit/Hyperactivity Disorder Susceptibility.

BACKGROUND: Genetic factors predispose individuals to attention-deficit/hyperactivity disorder (ADHD). Previous studies have reported linkage and association to ADHD of gene variants within ADGRL3. In this study, we functionally analyzed noncoding variants in this gene as likely pathological contributors. METHODS: In silico, in vitro, and in vivo approaches were used to identify and characterize evolutionary conserved elements within the ADGRL3 linkage region (~207 Kb). Family-based genetic analyses of 838 individuals (372 affected and 466 unaffected patients) identified ADHD-associated single nucleotide polymorphisms harbored in some of these conserved elements. Luciferase assays and zebrafish green fluorescent protein transgenesis tested conserved elements for transcriptional enhancer activity. Electromobility shift assays were used to verify transcription factor-binding disruption by ADHD risk alleles. RESULTS: An ultraconserved element was discovered (evolutionary conserved region 47) that functions as a transcriptional enhancer. A three-variant ADHD risk haplotype in evolutionary conserved region 47, formed by rs17226398, rs56038622, and rs2271338, reduced enhancer activity by 40% in neuroblastoma and astrocytoma cells (pBonferroni < .0001). This enhancer also drove green fluorescent protein expression in the zebrafish brain in a tissue-specific manner, sharing aspects of endogenous ADGRL3 expression. The rs2271338 risk allele disrupts binding of YY1 transcription factor, an important factor in the development and function of the central nervous system. Expression quantitative trait loci analysis of postmortem human brain tissues revealed an association between rs2271338 and reduced ADGRL3 expression in the thalamus. CONCLUSIONS: These results uncover the first functional evidence of common noncoding variants with potential implications for the pathology of ADHD.