FATP4 missense and nonsense mutations cause similar features in Ichthyosis Prematurity Syndrome

Patient material

We analyzed six cases with a typical IPS phenotype. The patients belongs to five families from Norway (IR85, IR125, and 25187), Denmark (DA1 comprising two affected siblings) and Iceland (25291; Table 1). No consanguinity was reported among parents and no families were inter-connected through the last four generations. The patients had a medical history with the clinical triad of premature birth, thick caseous desquamating epidermis, and neonathal asphyxia, typical for the IPS phenotype [11]. With the exception of Family DA1 (Figure 1a), no family history of IPS was recorded. Blood samples were obtained from probands and from healthy parents and siblings in three families (Family DA1, IR85 and IR125; Figure 1a).

Patient family ID	Exon	Nucleotide change	Amino-acid change	Zygosity	Origin
IR85	exon3 exon7	c.504c>a c.899a > g	p.C168X* p.Q300R*	compound heterozygous	Norway
IR125	exon2 exon3	c.103g>t c.504c > a	p.G35X p.C168X*	compound heterozygous	Norway
25187	exon3 exon10	c.504c>a c.1430t > a	p.C168X* p.V477D	compound heterozygous	Norway
25291	exon3	c.504c > a	p.C168X*	homozygous	Iceland
DA1	exon3 exon11	c.504c>a c.1511 g > a	p.C168X* p.R504H	compound heterozygous	Denmark

Family origin and zygosity of the identified mutations. The nucleotide and amino-acid change is indicated and the corresponding exon. *denotes previously published in Klar et al 2009.

Mutational analysis

Sequence analysis of the FATP4 gene was initially performed on the probands. In families DA1, IR85 and IR125, available parents and siblings were included for the investigation of mutational inheritance (Figure 1a). In total, 16 individuals (six patients and ten relatives) were included in the mutation analysis. DNA samples from 100 unrelated healthy Scandinavian control individuals were also analyzed to rule out polymorphic variants. Bidirectional sequence analysis of genomic DNA included all 13 exons of the FATP4 gene as well as intron-exon boundaries, 5' and 3' UTRs. Primer sequences used for amplification were designed with Primer 3 software (primer sequences available upon request). Sequencing was performed on an ABI 3730 DNA Analyzer (Applied Biosystems) using Big Dye Terminator v3.1 Cycle Sequencing Chemistry (Applied Biosystems) according to protocols recommended by the manufacturer. Base calling was performed with Sequencing Analysis v 5.2 (Applied Biosystems) and the sequences were analyzed with the Sequencher software v4.1 (Gene Codes Corporation).

Structural modelling

To analyze the degree of conservation of the predicted amino acid substitutions, multiple sequence alignment was performed using ClustalW [12]. The human (Homo sapiens) FATP4 protein sequence was compared to mouse (Mus musculus) and dog (Canis familiaris) as well as the other members of the FATP protein family (FATP1, FATP2, FATP3, FATP5 and FATP6)

Mutational analysis

Sequence analysis of the FATP4 gene revealed two sequence variants predicted to be associated with IPS in each proband. In total five different mutations were identified (Table 1). Five probands were compound heterozygous for the previously reported c.504c > a transition in exon 3. This variant predicts the previously reported p.C168X nonsense mutation. One proband from the Icelandic family 25291 was found homozygous for this mutation. We also identified a novel c.103g > t transition in exon 2. This variant results in a p.G35X nonsense mutation (Figure 1b) and an early truncation of the protein. Another three sequence variants were predicted to result in missense mutations. The p.Q300R is caused by an a to g transition at nucleotide 899 (c.899g > a) in exon 7 and results in the substitution of the polar uncharged hydrophilic amino acid glutamine for the positively charged hydrophilic arginine. This mutation has been reported previously [2]. The novel p.V477 D is caused by a t to a transition at nucleotide 1430 (c.1430t > a) in exon 10 and results in the change of the nonpolar hydrophobic amino acid valine to the negatively charged hydrophilic aspartic acid. The p.R504 H mutation is another novel variant caused by a g to a transition at nucleotide 1511 (c.1511g > t) in exon 11 and results in the substitution of the positively charged hydrophilic arginine for the similarly positively charged hydrophilic histidine (Table 1). Healthy family members were available in family IR85, IR125 and DA1 and the analysis showed that the mutations segregated with the disease with parents being heterozygous carriers for the respective mutations (Figure 1a). The three missense mutations (p.Q300R, p.V477 D and p.R504H) were situated in the highly conserved AMP-binding domain (aa 103-536). The p.Q300R is situated in the AMP/AMP motif involved in ATP binding and adenylate formation, and the novel p.R504 H is located in the FATP motif important for fatty acid binding (Figure 1c). All variants were excluded as common variants or polymorphisms by comparing to databases (dbSNP 131) and by the analysis of 200 Scandinavian control chromosomes. Thus, among five different IPS associated mutations identified in the five families, the p.G35X, p.V477 D and p.R504 H mutations are novel whereas the two mutations (p.C168X and p.Q300R) were described previously [2].

Structural modelling

Multiple sequence alignment of the FATP4 protein was performed in order to estimate inter-species conservation of certain segments and to identify regions shared with other members of the FATP family of proteins. Alignment of the FATP4 protein sequence from human (Homo sapiens), mouse (Mus musculus) and dog (Canis familiaris) demonstrated strong conservation of the three amino acids corresponding to the missense mutations in the patients (Figure 1d). Alignment of the FATP4 sequence to other members of the FATP family of proteins showed complete identity of the amino acids corresponding to the novel missense mutations, p.V477 and p.R504 (Figure 1e). The previously identified missense mutation, p.Q300R, is situated in a protein domain shared exclusively with FATP1.