Written informed consent was obtained from the patient's legal guardians. Molecular analysis was performed when he was 6 years old during the mother's pregnancy with her second baby. Further molecular genetic testing of the parents was performed to assess whether both variants were on the same (cis) or a different (trans) TBX19 allele and the abortion material was also analyzed molecularly. The pedigree was shown in Figure 1A.
In the molecular analysis of the index case, a novel heterozygous c.665delG (p.Arg222Lys*4) variation in exon 4 (Figures 1B,C) and pathologic heterozygous c.856C>T (p.Arg286Ter) variation in exon 6 (Figure 1D) of the TBX19 gene (NM_005149.2, NP_005140.1) were detected in the compound heterozygous state. The initial genetic test suggested a possible splice site variation (c.665 + 1delG) in intron 4 of the TBX19 gene instead of a c.665delG variant, because of the consecutive GG repeat at the exon-intron boundary. Therefore, further molecular analyses were performed at the mRNA level to gather information about the location of the variation (the last nucleotide of exon 4 or the first nucleotide of intron 4) and to identify a possible splicing effect. The mRNA analyses revealed that the real variation was c.665delG (p.Arg222Lysfs*4) and that it was a novel exonic frameshift mutation in spite of the splice site mutation (Figure 1C). As a consequence of the deletion of guanine 665 at the cDNA level, the first affected amino acid arginine was replaced with lysine at residue 222, and the new reading frame ended with a stop codon at position 4.
The other c.856C>T substitution was a nonsense variant predicted to result in the substitution of an arginine by a premature stop codon at position 286 in the protein (p.Arg286Ter) (Figure 1D). The c.856C>T variant was previously described in the Human Genome Mutation Database; it is associated with isolated deficiency of the pituitary and ACTH (HGMD #CM014746). Also, it is listed as a pathogenic allele in the dbSNP (rs74315376) and ClinVar databases,1 with a lower minor allele frequency of 0.00005 (6 of 121,334 alleles).2
Molecular analysis revealed that the mother was a carrier of the c.665delG variation (Figures 1B,C) and the father was a carrier of the p.R286X mutation (Figure 1D). The abortion material also showed the same pathological variations in the TBX19 gene.