Sobol M. Genetic and molecular studies of Ichthyosis Prematurity Syndrome and Isolated Nail Dysplasia

Genetic basics of two ectodermal disorders - Ichthyosis Prematurity Syndrome and Isolated Nail Dysplasia - are presented in this thesis. Ichthyosis prematurity syndrome (IPS) is a rare form of autosomal recessive ichthyosis characterized by polyhydramnion and premature birth of the affected child. Newborns exhibit respiratory complications and a thick caseous desquamating epidermis. During the first months of life the symptoms become gradually milder. Patients suffer from a lifelong non-scaly ichthyosis with atopic manifestations. Histological analysis of the skin reveal thickening of the epidermis. Patients with IPS have been reported from Scandinavia, Middle East, southern Europe and Africa. The disease is associated with mutations in the fatty acid transport protein 4 (FATP4) gene. The FATP4 protein plays a central role in the transport and activation of fatty acids in the epidermis for normal epidermal barrier function. Eight distinct FATP4 mutations have been reported in IPS patients. The p.C168X nonsense variant is prevalent in the Scandinavian IPS population and all patients from this geographical region were found homozygous or compound heterozygous for this mutation. Herein the identification of FATP4 mutations in six Scandinavian probands including three novel mutations is reported. All six patients were compound heterozygous or homozygous for the p.C168X mutation which further illustrates the high prevalence for this mutation in the northern European population. Also a novel p.G35X nonsense mutation was identified. Another three sequence variants were predicted to result in missense mutations. p.Q300R missense mutation has been reported previously. There were two novel messense mutations p.V477D and p.R504H identified in present study. All mutations were inherited by probands from healthy parents who were heterozygous carriers of different mutations. All mutations were located in the functional domains of FATP4 protein. The three missense mutations (p.Q300R, p.V477 D and p.R504H) were situated in the highly conserved AMP-binding domain. Strong conservation of the amino acids corresponding to the novel missense mutations in the patients had been observed. There appear to be no obvious correlations between the nature of mutations in the FATP4 protein and the IPS phenotype. This suggests that the missense mutations reported herein have deleterious effects on FATP4 function which are similar to the effects of the nonsense mutations. This study adds to the mutational spectrum associated with IPS, which may improve genetic diagnosis of the disease as well as future functional analysis of FATP4. Isolated Nail Dysplasia (IND) is a rare congenital disorder. All affected individuals show a variable degree of onychauxis (thick nails), hyponychia, and onycholysis of fingernails and toenails. Fingernails had a claw-like appearance. No other disorders or anomalies of ectodermal tissues (i.e., hair, teeth, sweat glands, or skin) were noted and individuals with dysplastic nails had normal hearing, normal psychomotor development and reported normal sweating as well as normal hair growth. Two consanguineous Pakistani families (F1 and F2) affected by autosomal-recessive IND were investigated. The affected individuals from family F1 presented with a more severe nail dysplasia compared to affected individuals from family F2. 800 kb region on chromosome 8 appeared to be the locus-candidate for the disorder. Sequencing analysis of FZD6 revealed two different mutations in the patients: p.E584X and p.R511C. FZD6 belongs to the heptahelical class of FZD receptors with an internal PDZ-interacting motif necessary for the recruitment of the phosphoproteins DVL 1-3 and other signaling factors as well as for trafficking of the receptor. Both FZD6 mutations result in alterations of the intracellular tail in the key functional PDZ-domain. FZD6 is an important protein for several WNT-signaling pathways that are indispensable for numerous developmental processes such as tissue morphogenesis, differentiation, and regeneration in all animals. By interaction with different WNT and DVL proteins it activates number of cellular processes. The nonsense mutant fibroblasts expressed 13% of FZD6 mRNA levels in primary control fibroblasts supporting nonsense-mediated mRNA decay. The interaction between FZD6-nonsense and any of DVL proteins were not detected that indicates the loss of main functional activity in FZD6 with nonsense mutation. FZD6 with the missense mutation showed improper integration into the cell membrane, increased lysosomal degradation that result in reduced capability for DVL recruitment. The investigation of downstream effects of WNT-3A and WNT-5A stimulation of nonsense-mutated fibroblasts was performed by analysis of levels of mRNA expression for B-catenin, DKK1 and MSX1. Increased mRNA levels were observed in WNT-5A-stimulated control fibroblasts but not in patient fibroblasts for all three factors. These results suggest that patient fibroblasts homozygous for the FZD6 nonsense mutation fail to respond properly to both WNT-3A and WNT-5A activation. Present combined results show that IND can be caused by dysfunctional FZD6 or loss of FZD6 with a subsequent misregulation of several FZD6-mediated pathways required for proper formation and regeneration of nails throughout life.