Novel hereditary spherocytosis-associated splice site mutation in the ANK1 gene caused by parental gonosomal mosaicism

Abstract

Hereditary spherocytosis (HS) is a heterogeneous condition of inherited hemolytic anemia characterized by anemia, jaundice, cholelithiasis and splenomegaly with a prevalence of 1 in 10,000 in China 1 . Diagnosis of HS is mainly based on a positive familial history, clinical features and laboratory data, and observation of spherocytes in a peripheral blood smear. -thalassemia genetic mutation screen found no abnormality. Osmotic fragility was increased, and spherocytes were observed in a peripheral blood smear. Both her 28-year-old father and 27-year-old mother were asymptomatic.The available laboratory data are summarized in Table 1.Targeted NGS was performed for DNA extracted from peripheral blood mononuclear cells (PBMCs) of the proband using a panel targeting all exons and adjacent introns of ANK1, EPB42, SLC4A1, SPTA1, and SPTB genes. For the proband, the mean depth was 2,096-fold; 99.00% of the mapped reads were on target, and 98.41% of the target bases were covered at least 20 times. The mean uniformity of base coverage was 95.07%. 97 variants were identified in the proband, distributed in exons, introns, 3′-UTR, 5′-UTR and splice site. These variants were filtered according to mutation type, amino acid alteration, effect on reading frame, minor allele frequency in 1000G, ExAC, dbSNP, ClinVar, and gnomAD databases, records in the HGMD database, and mutation functional prediction. After filtration, a novel heterozygous ANK1 c.3084-2A>G (NM_001142446.1) splice site mutation (covered by 1286-fold) was selected for further validation by Sanger sequencing, and the ANK1 c.3084-2A>G mutation was not found in the 1000G, dbSNP, ClinVar, ExAC, gnomAD, or HGMD databases. Sanger sequencing confirmed the heterozygous ANK1 c.3084-2A>G mutation in the proband. Genetic screening within the family members revealed that this mutation was absent from her mother. However, her father's chromatogram showed a small peak of G within the A reference nucleotide at the ANK1 c.3084-2 position, which was repeated in a buccal swab sample and was more obvious in his sperm (Figure 1). These results suggest the probability of germline and somatic mosaicism for the proband's father. To evaluate this hypothesis, NGS was further performed for DNA from PBMCs, the buccal swab, and the sperm of her father. The ANK1 c.3084-2A>G mutation (chr8:41552851) was present at 16%, 15%, and 29% with 735-, 745-, and 901-fold sequence coverage for PBMCs, the buccal swab, and sperm, respectively, providing evidence of germline and somatic mosaicism. Buccal mucosa may contain variable contamination of leukocytes. Taken together, her father was a combination of germline and somatic mosaicism, and the proband's constitutional ANK1 c.3084-2A>G mutation was caused by her father's mutant germline cells.To explore the splice site effect of the ANK1 c.3084-2A>G mutation (located in intron 27), mRNA of PBMCs was isolated from the family members and reverse transcribed into cDNA. Primers covering exon 27 to exon 29 to generate a 624 nucleotide PCR product were used. A...

Genetic and Clinical Characteristics of Patients With Hereditary Spherocytosis in Hubei Province of China

“…Sanger sequencing of the reverse transcribed cDNA from peripheral blood is a common and easy method to validate the effect of splice site variants on mRNA splicing ( Wang X. et al, 2018 ). However, in this study, this method failed to detect abnormally spliced transcripts in two families carrying splice site variants.…”

“…Targeted NGS was performed as previously described ( Wang X. et al, 2018 ). Genomic DNA was extracted from peripheral blood with the PANA9600 Automated Nucleic Acid Extraction System (Tianlong, Xi’an, China).…”

Advances in understanding the pathogenesis of red cell membrane disorders

“…The inheritance pattern is dominant in approximately 75% of cases, and non‐dominant (ndHS) in the remaining 25% of patients (Andolfo et al , ; Narla & Mohandas, ). The latter group comprises patients with a recessive inheritance pattern due to biallelic mutations in SPTA1 and EPB42 , and patients with de novo events that are mostly related to haploinsufficient mutations in the ANK1 and SPTB genes (Wang et al , ). However, ANK1 missense variants are common in recessive HS, whereas ANK1 and SLC4A1 null variants prevailed in dominant HS (Eber et al , ).…”

Whole exome sequencing identified a novel mutation (p.Ala1884Pro) of β‐spectrin in a Chinese family with hereditary spherocytosis

“…Hereditary spherocytosis (HS) is a genetic disease of the red blood cell (RBC) membrane, characterized by anemia, jaundice and splenomegaly . In the peripheral blood smear experiment, the RBCs of HS patients may be present as spherical‐shaped cells . In the clinic, the signs and symptoms of HS can range from mild to severe and patients with a severe situation of HS may demonstrate short stature, delayed puberty and skeletal abnormalities .…”