Hennekam Syndrome Can Be Caused by FAT4 Mutations and Be Allelic to Van Maldergem Syndrome

Supplementary data

Hennekam syndrome can be caused by FAT4 mutations and be allelic to Van Maldergem syndrome

Marielle Alders, Lihadh Al-Gazali, Isabel Cordeiro, Bruno Dallapiccola, Livia Garavelli, Beyhan Tuysuz, Faranak Salehi, Martin A. Haagmans, Olaf R. Mook, Charles B. Majoie, Marcel M. Mannens & Raoul C. Hennekam

Homozygosity mapping

Homozgosity mapping in family F1 (F1-1, F1-2 and F1-3) was performed using the Affymetrix® Genome-Wide Human SNP Array 6.0 array. Results were analyzed using Nexus Copy Number™ software version 7 (BioDiscovery). Homozygous regions of more than 5 Mb were called (SNPRank Segmentation). A shared homozygous region was present on chromosome 4q27-q28.3 (Table S1). Analysis of the genotypes showed that all three sibs shared a genotypically identical homozygous region between rs684552 (g:122146514) and rs1380631 (g:137687991).

Table S1. Homozygous regions F1-1, F1-2 and F1-3.

Figure S1. Electropherograms showing mutations in FAT4.

Figure S2. Evolutionary conservation FAT4 missense mutations.

p.Ser4284Phe

Analysis splice site intron 7 – exon 8 FAT4

Patient F4 carried an intronic mutation at position c.7200-8A>C according to the annotation inNM_015284.3. Comparison of the human FAT4 protein to the homologous gene in other species showed that the human protein lacks two aminoacids (Supplementary figure S4a). To further analyze the splicing position at the splice acceptor site of intron 7 RT PCR was performed. mRNA was extracted from human umbilical cord using TRIzol® Reagent (Life Technologies) according to the manufacturer’s protocol. In addition, mRNA extracted from brain tissue (hippocampus) was commercially obtained from BD. Reverse transcription was done with Superscript II (Invitrogen) and random hexamers according to the manufacturer’s protocol. Amplification of FAT4 cDNA using primers in exon 7 and 8 and subsequent sequencing showed that in both tissues splicing occurred 6 nucleotides upstream of the annotated intron 7 in the human reference sequence (NM_024582.4), thus at Chr4:g.126367447-126367448 instead of Chr4:g.126367453-126367454 (hg19). (Supplementary figure 4b). This transcript has been assigned refseq nr NM_001291303.1. This result places the mutation identified in patient F4 in the invariant splicing nucleotides of intron 7/exon 8 splicing (c.7200-2A>C) and most likely disturbed normal splicing at this site.

Supplementary Figure S3. Evidence for alternative splice site of intron 7/exon 8.

Figure S3. Evidence for usage of alternative splice site of intron 7/exon 8. S3a. Alignment of human, mouse, rat and zebrafish FAT4 shows a deletion of two aminoacids in the refseq human FAT4 protein NM_015284.3. S3b. RTPCR and sequencing shows splicing of FAT4 occurs 6 nt upstream of the annotated splice site in NM_015284.3 in hippocampus brain and umbilical cord (UC) RNA (Genbank accession numbers KJ131481 and KJ131482) .