Discussion
ANK1 Mutation is the most common cause of HS. ANK1 mutations is inherited in both AD (autosomal dominant) and AR (autosomal recessive) patterns in HS and the majority is de novo mutation [1]. These mutations locate the entire ANK1 gene, throughout the promoter region and coding exons [12, 13]. To date, a total of 62 mutations are included in the HGMD public database. Missense/nonsense and small deletions have been identified in approximately 70% of all mutations in the ANK1 gene. Seven splicing mutations, occurring in IVS1,16,20,22,28 and 38, are included in the HGMD database. In this study, we described a Chinese family with a proband and his mother affected by HS. A de novo mutation (c.856C>T p.R286*) causing a premature stop codon in exon 9 of ANK1 was found by investigating this family through NGS followed by Sanger sequencing to certify the relationship between the ANK1 mutation and HS.
ANK1 plays a pivotal role in the stabilization of the membrane, providing the main membrane binding site for the spectrin-based membrane skeleton. ANK1 consists of three structural domains: a multiple repeats N-terminal domain(89kD), a spectrin-binding center region(62kD) and a regulatory C-terminal domain(55kD) [7, 14, 15]. Mutations in the spectrin-binding domain and regulatory C-terminal domains result in the most severe anemia compared with those located in the other domains [14, 16]. In this family, a ANK1 c.856C>T mutation occurred in the N-terminus region. This mutation was not found in the gnomAD ,1000G, ExAC and HGMD database, and confirmed as a novel mutation. According to bioinformatics analyses, this point mutation generates a premature translation termination codon resulting in a truncated ANK1 protein, losing the important spectrin-binding and regulatory C-terminal domains and leaving only partial of the N-domain, which might cause HS. Moreover, consistent with the genetic diagnosis, the symptom of the mother was ameliorated after splenectomy, further supporting the genotype-phenotype relationship.
NGS provides a comprehensive and cost-effective approach to molecular diagnosis of hereditary hemolytic anemia, especially in cases where biochemical testing is unreliable due to multiple transfusions. In previous research [17], a female individual presented with a yellow complexion, jaundice and splenomegaly without other pathological symptoms or signs on the second day after birth. And then the girl who was clinically diagnosed with HS and under laparoscopic splenectomy because of splenomegaly and hemolysis when she was 6-year-old. Finally, using NGS detected a de novo nonsense ANK1 mutation (c.796G > T, p. Glu266X), which caused a substitution from glutamic acid to a premature stop at codon 266 and may lead to HS. But no mutation was detected in her parents. Thus, it is efficient and important for us to using NGS to identify inherited, rare gene mutations which are associated with a high risk for the development of diseases. If diagnosed earlier, fewer newborn infants would develop severe anemia and hazardous hyperbilirubinemia and it would remind pediatricians to closely monitoring of infants with HS during the first 6 months of life that is important for appropriate clinical management.
In summary, our results demonstrated an ANK1 c.856C>T mutation may lead to a premature stop at codon 286 and may be responsible for HS in two patients from a Chinese family. Identifying the underlying genetic cause not only helps with the management of the patients, it also facilitates accurate genetic counselling and helps in guidance regarding the predicted severity of clinical phenotype.