An SP-B Gene Mutation Responsible for SP-B Deficiency in Fatal Congenital Alveolar Proteinosis: Evidence for a Mutation Hotspot in Exon 4

Abstract Mutations and polymorphisms within the human SP-B locus have been linked to fatal congenital alveolar proteinosis (CAP) and associated with respiratory distress syndrome (RDS), respectively. In the present study we used PCR and direct sequence analysis of the SP-B gene of three individuals from a family with CAP to search for additional SP-B mutations resulting in CAP and/or polymorphisms that could be used as markers in association studies of RDS and/or CAP. We found three novel mutations/polymorphisms in this family. One is a C/A substitution at nt 1013 at the splice junction of intron 2-exon 3. A second one is a single base T deletion at nt 1553 in exon 4. The single base (T) deletion at nucleotide 1553 (1553delT) shifts the reading frame at amino acid 122 (122delT) and creates a premature termination codon at amino acid 214 in exon 6. The mutated gene produces no mature SP-B protein. Genotype analysis from the nuclear family carrying this mutation showed that both parents and three of the four living children are heterozygous for the mutation. One of the four living children is homozygous for the normal allele and a child that died in the perinatal period from CAP is homozygous for the mutation. A third change is a C/T substitution at nt 1580 in exon 4 that changes amino acid 131 from threonine to isoleucine (Thr131Ile). The location of a previously reported mutation, 121ins2 (1), is only 4 nt upstream of 122delT, and the missense mutation Thr131Ile (exon 4) is only 27 nt downstream of 122delT. These changes are within or in close proximity to a CCTG sequence and a poly(C) tract, both of which are shown in other systems to be mutation hotspots. The 122delT occurs within the CCTG and the poly(C) tract sequences, the Thr131Ile occurs 26 nt downstream from the CCTG sequence, and the 121ins2 occurs 2 nt upstream from CCTG sequence and within the poly(C) tract. The present observations suggest that the short SP-B sequence containing the CCTG motif and the poly(C) tract is a mutation hotspot.