Identification by whole-exome sequencing of novel mutation c.64C > G in the BTK gene of a fetus with X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) or Bruton’s disease is a rare genetic disorder, discovered in 1952, that mitigates the immune response to infections1. Early studies have associated mutations in the Bruton tyrosine kinase (BTK) gene (GDB 120542) with XLA development2,3. The BTK gene, spanning 37.5 kilobases (kb) with 19 exons, is located on the X chromosome at Xq21.3–Xq22 and encodes Bruton agammaglobulinemia tyrosine kinase (Btk), a 77-kiloDalton (kDa) protein. Btk plays an essential role in the maturation of B cells within the bone marrow. Its mutation often stalls differentiation of immature pro-B into pre-B lymphocytes, decreasing the number of peripheral mature B cells4. We report the identification of a new mutation in a fetus with XLA, identified prenatally by whole-exome sequencing, predicted to cause a drastic alteration in BTK gene function. A 26-year-old primigravida was referred to our center after detection by ultrasound screening at 25+6 weeks’ gestation of fetal brain abnormalities (ventriculomegaly, cerebral calcification and intracranial space-occupying lesion) (Figures 1a and b), which were confirmed by magnetic resonance imaging at 28 weeks (Figures 1c and d). Lymphocyte immunophenotyping of the fetus by flow cytometry at 28 weeks showed that 97.6% of its lymphocytes were CD3+ (T-cells) (Figure S1), but B-cell and NK-cell levels were almost undetectable. Peripheral venous blood of the mother and umbilical cord blood from a normal pregnancy were used as controls. Both samples were found to have normal lymphocyte counts (Figure S1). Exome sequencing of the fetal BTK gene revealed a homozygous missense mutation, c.64C>G, caused by substitution of a single amino acid (p.Pro22Ala) in the Btk protein (Figure S2). This mutation has not yet been identified in the BTK database (http://structure.bmc.lu.se/ idbase/BTKbase/). Exome sequencing of the mother found her to be a carrier of the mutation. To confirm further the presence of this mutation, we amplified the DNA fragment that contained the mutation by polymerase chain reaction (PCR) and subsequently performed Sanger sequencing on the PCR product. In addition, the DNA fragment from 50 healthy donors was amplified and sequenced and no c.64C>G mutations were detected in these cases. This finding implicated the c.64C>G mutation as a novel XLA-relevant mutation. We have shown a novel mutation in the BTK gene of a fetus with a severe phenotype, including B-cell deficiency. The central message of this case is to emphasize the contribution of whole-exome sequencing in the diagnosis of complex human disease, especially in the context of prenatal screening. In addition, the BTK gene mutation demonstrated in this study expanded the spectrum of known XLA-causing mutations.