An intronic alteration of the fibroblast growth factor 10 gene causing ALSG-(aplasia of lacrimal and salivary glands) syndrome

Abstract

Background: A combined aplasia, hypoplasia or atresia of lacrimal points and salivary glands is rarely diagnosed. Those patients suffer from epiphora, xerostomia and severe dental caries. This phenotype represents the autosomal-dominant aplasia of lacrimal and salivary glands syndrome (ALSG). Recently, aberrations of the Fibroblast Growth Factor 10 (FGF10) gene have been identified to be causative for this disorder.

Methods: We performed a sequence analysis of the FGF10 gene of a patient with ALSG-syndrome and his also affected brother as well as 193 controls. The FGF10 transcript was analyzed using RNA extracted from primary fibroblasts of the patient's mucosa.

Results: We detected a novel heterozygous sequence variation in intron 2 (c.430-1, G > A) causing the ALSG syndrome. The alteration derogates the regular splice acceptor site and leads to the use of a new splice acceptor site 127 bp upstream of exon 3. The aberration was detected in the genomic DNA derived from two affected brothers, but not in 193 control individuals. Furthermore, no diseased member of the family displayed additional abnormalities that are indicative for the clinically overlapping lacrimo-auriculo-dento-digital syndrome (LADD).

Conclusion: This family-based approach revealed an intronic variation of the FGF10 gene causing ALSG-syndrome. Our results expand the mutational and clinical spectrum of the ALSG syndrome.

Figure 1

Computer tomography scans demonstrating the pathological findings of an ALSG patient. The pathological areas are marked. 1a: The patient shows an aplasia of both lacrimal punctae associated with a mucocele of the lacrimal sac imposing as a benign soft tissue tumor in the region of the medial canthus. 1b: Prosthetically (gold cap) treated teeth after caries-related destruction, especially in the molar region. 1c-d: Axial (c) and coronal (d) computer tomography scan demonstrating mucoceles originating from the lacrimal sac. The os lacrimale is partly destroyed, on the right side more pronounced than on the left side. 1e: Axial computer tomography scan showing a total aplasia of both parotid glands.

Figure 2

a: A heterozygous sequence variation in the terminal nucleotide of intron 2 of the FGF10 gene (g.85478 corresponding to c.430-1, G > A) was detected which alters the consensus motif for splice acceptor site recognition. The upper panel shows a representative electropherogram of a healthy individual. The middle and the lower panel demonstrate the sequence variation in genomic DNA derived from the patient and his brother. The changed nucleotide is marked by an arrow. 2b: Chart showing the exon/intron structure of the FGF10 gene. The characteristic polypyrimidine stretch and the consensus acceptor site are shown. The changed nucleotide is marked by an arrow.

Figure 3

Analysis of FGF10 transcript applying long range RT-PCR. In order to analyze for transcripts that may include the entire intron 2, a long range RT-PCR protocol was established that works to amplify the 6.2 kb-fragment spanning from exon 2 to exon 3 using gDNA as template. Applying the optimized conditions to amplify the FGF10 transcript, no accordant PCR product was present. 1, 3: patient; 2, 4: control; MW: molecular weight marker.

Figure 4

Detection and analysis of an aberrant transcript. 4a: Separation of FGF10 RT-PCR amplificates using sensitive silver-stained polyacrylamid gels reveals additional fragments for the patient's samples but not for control samples. Upper panel, Amplification of the FGF10 transcript using oligonucleotides designed to amplify exon 1 to exon 3 sequences; Lower panel, Amplification of the FGF10 transcript using oligonucleotides designed to amplify exon 2 to exon 3 sequences. C: control; P: patient. 4b: Sequence analysis of the excised and re-amplified additional exon 1/exon 3 fragment. The c.430-1, G > A aberration results in the use of an alternative splice acceptor site 127 bp upstream of exon 3. 4c: Putative effect of the 127 bp insertion on protein translation.