Utility of long-read sequencing to delineate a rare large deletion of beta-globin gene which escaped Sanger sequencing at prenatal diagnosis in a family clustered with hereditary persistence of fetal hemoglobin.

OBJECTIVE: Thalassemias, including alpha and beta thalassemia, are the most common monogenic inherited Mendelian disorder in Taiwan. The mutation spectrums of alpha and beta thalassemia are well known in the Taiwanese population. Among them, mutations with the format of large deletions are more prevalent in alpha thalassemia whereas point mutations or small insertion/deletions (indels) are much more prevalent in beta thalassemia. Hereditary persistence of fetal hemoglobin (HPFH), a clinical subtype of beta thalassemia, is a relative uncommon condition during prenatal diagnosis in the Taiwanese population, most often caused by large deletions and may escape diagnostic panels based on Sanger sequencing. This study aimed to explore the feasibility of third-generation long read sequencing (LRS) in the genetic diagnosis of such rare condition in a prenatal setting.

CASE REPORT: A woman at her second trimester pregnancy went to our hospital due to a high suspicion was impressed that both she and her husband were carriers of beta thalassemia by their primary care obstetricians. Due to time constraint in a prenatal setting, the blood samples of both couple and the fetal sample (obtained through amniocentesis) were simultaneously taken and genotyping of beta-globin gene was performed in the family trio. Beta-globin gene sequencing together with karyotyping and chromosome microarray were conducted in the fetal sample (amniotic fluid). Initial results by Sanger sequencing-based molecular diagnostics for beta-globin gene only depict the maternal mutation allele whereas a large deletion of the beta-globin gene may be hidden in the fetus and the father with unknown size was suspected from the chromosome microarray result. LRS therefore was performed and a very rare 27411-bp deletion was noted both in the fetus and the father, and further confirmed by the methodologies based upon polymerase chain reaction (PCR).

CONCLUSION: LRS can aid in the prenatal diagnosis of rare conditions like HPFH, given the current practice that Sanger sequencing-based molecular diagnostic platform is utilized here in Taiwan as first-line for beta thalassemia. Meanwhile, high resolution chromosome microarray, like the oligonucleotide array comparative genomic hybridization in this study, can also aid in the genetic diagnosis of deletion-type beta thalassemia, despite the actual size and breakpoints of the deletion may only be delineated by LRS in a straight-forward fashion, and can be further verified with traditional PCR-based methodologies.