Introduction

Ichthyosis vulgaris (IV) is a common disorder with a reported incidence of 1 in 250 individuals based on a survey of 6,051 healthy English schoolchildren (Wells and Kerr, 1966). In the Chinese population, the prevalence rate is even higher, reaching about 2.29% (Lei et al., 1992). IV is characterized by palmar hyperlinearity, keratosis pilaris, and a fine white scale over the exterior surface of the lower abdomen, arms, and legs. The cheeks and forehead may also be affected in early childhood, but it usually diminishes with age (Shwayder and Ott, 1991). IV is also associated with atopic manifestations, including asthma, eczema, and hay fever (Ziprkowski and Feinstein, 1972). The disease can start early at the age of 3 months, although it becomes apparent by 5 years of age in the majority of cases (Okulicz and Schwartz, 2003).

Genetically, IV is inherited in an autosomal-dominant manner (Wells and Kerr, 1966). An IV gene was mapped to chromosome 1q22 in an American family (Compton et al., 2002) and two Chinese families (Zhong et al., 2003). Recently, the IV gene at the 1q22 locus was identified as the filaggrin gene (FLG), which encodes a keratin filament-aggregating protein critical to epidermal barrier formation and hydration (Smith et al., 2006; Nomura et al., 2007; Sandilands et al., 2007).

In this study, we characterized two Chinese families with autosomal-dominant IV and clinically similar features. Both families were not linked to the FLG gene. A genome-wide scan in the two families identified a previously unreported IV locus on chromosome 10q22.3–q24.2. This is the second locus identified for IV.

Results and Discussion

We studied two Chinese families with a similar phenotype of typical clinical features of IV. The age of onset in the two families was very early: 1 month for the proband in the first family (family 1) and a few days after birth for the proband in the second family (family 2). The patients in both families displayed fine, white scaling over the exterior surface of the extremities (for an example, see Figure 1), marked palmar hyperlinearity of palms and soles, and common dry skin. Mild keratosis pilaris was present on the exterior surface of the extremities. IV features were visible from the neck to the ankles in some patients. The big flexures were spared. Nails, hairs, and teeth appeared normal. The patients sweated normally and did not display hypohidrosis. The IgE level was measured in two patients in family 1, the probands IV:2 and III:3 (Figure 2a), and appeared to be normal (121.26 and 91.89 IU ml^{- 1}, respectively). The patients did not exhibit any atopic manifestation, and thus are different from the families with FLG mutations. Also different from families with the FLG mutations, the two Chinese families in this study showed complete penetrance of the IV phenotype, which may be due to the early onset of the disease in the families. These results imply that there may be a previously unreported disease gene in the two Chinese IV families. The pedigree structure for the two families is shown in Figure 2. The inheritance pattern in the two families appeared to be autosomal dominant.

Figure 1.

Typical clinical features of IV in Chinese family 1. Note the marked, fine, white flaky scaling on the exterior surface of the dorsum of the pretibial regions.

Full figure and legend (50K)

Figure 2.

Exclusion of linkage to the FLG gene on chromosome 1q in two Chinese families with IV. (a) The pedigree structure for family 1 and genotyping results for four markers on chromosome 1q22. Affected individuals are shown as filled squares (males) or circles (females), and normal individuals are indicated with open symbols. Deceased individuals are marked with slashes (/). The proband is indicated by an arrow. Haplotypes are shown with vertical bars, disease-linked haplotype with filled bars, and normal haplotypes with open bars. Positions for recombinations are marked with arrowheads. (b) The pedigree structure for family 2, and linkage and haplotype analyses in the family. (c) Ideogram of chromosome 1 showing the position and distance of FLG to its flanking markers.

Full figure and legend (114K)

Our initial linkage analysis was focused on the FLG gene on chromosome 1q22 as well as the chromosomal regions or genes identified for ichthyosis-related diseases. No linkage was found with the FLG gene as the two markers flanking the FLG gene, D1S498 and D1S1595, generated LOD scores of - 6.26 and - 6.30 in family 1, and - 3.30 and - 1.13 in family 2, respectively (Table 1). Haplotype analysis with multiple markers flanking the FLG gene, including D1S442, D1S498, D1S1595, D1S2721, and D1S2635, identified multiple obligate recombinants (patients II:4, III:11, and IV:5 in family 1 and III:2 in family 2) and one non-obligate recombinant, III:1 in family 2 (Figure 2). These results strongly suggest that the FLG gene is unlikely to be responsible for IV in the two Chinese families. Negative LOD scores also ruled out linkage of the two Chinese IV families to other known autosomal ichthyosis-related genes, including ABCA12, GJB2, ich, KRT1, KRT10, KRT2A, TGM1, CLDN1, LI5, LI3, INLNE, STS G4523, and STSLC-1 (Table 1). These results excluded the previously reported genes and linkages for IV and ichthyosis-like diseases in the two Chinese IV families and suggest that the families may be potentially associated with a previously unreported IV gene.

Table 1 - Two-point LOD scores for markers spanning known autosomal-dominant IV and ichthyosis-associated genes in the two Chinese families.

Full table

To identify the chromosomal location of a new gene responsible for IV, we undertook a genome-wide linkage scan in the larger family, family 1, which was followed by fine mapping. LOD scores for all genotyped markers were plotted against the physical position of each marker in Figure S1. Positive linkage was identified only with markers on chromosome 10q22.3–q24.2. No other markers showed a LOD score of >1.5. The LOD scores for the chromosome 10 markers studied are shown in Table 2. Three markers, D10S1717, D10S1765, and D10S1680, generated LOD scores of >3, the cutoff LOD score for significant linkage. These results suggest that there is a previously unreported genetic locus for IV on the long arm of chromosome 10 (Figure 3).

Figure 3.

Identification of a new genetic locus for IV with markers on chromosome 10q22.3–q24.2. (a) Linkage and haplotype analyses in family 1. (b) Linkage and haplotype analyses in family 2. (c) Ideogram of chromosome 10 showing Giemsa banding patterns and the detailed location of the new genetic locus for IV.

Full figure and legend (144K)

Table 2 - Two-point LOD scores for markers on chromosome 10q22.3–q24.2 at different recombination fractions () in the two Chinese IV families.

Full table

Haplotype analysis was carried out to analyze the recombination events in family 1 (Figure 3a). Normal individual III:7 showed a recombination event between markers D10S569 and D10S201. Normal individual IV:8 displayed a recombination event between markers D10S571 and D10S1709. These results suggest that the gene responsible for IV in family 1 lie between D10S569 and D10S1709 on chromosome 10q22.3–q24.2, a genomic region of 20.7 cM or 20.3 Mb (Figure 3c).

Family 2 was genotyped with nine markers at the newly identified IV locus in family 1. As shown in Figure 3b, all nine markers showed positive linkage with IV. The results from haplotype analysis are shown in Figure 3b, and the LOD scores are shown in Table 2. These results provide further support that a new IV gene is located on chromosome 10q22.3–q24.2. It is interesting to note that although the two families are linked to the same 10q IV locus, they do not share the same haplotype (Figure 3), raising a possibility that the two families may carry different mutations in the IV gene at this locus. The combined LOD scores for the two families are shown in Table 2. The combined maximum LOD score was 3.95 for marker D10S1717 and >3.0 for seven other linked markers (D10S201, D10S1717, D10S1744, D10S1765, D10S185, D10S1680, and D1S0S571) (Table 2).

There are over 84 known genes and 30 putative genes at the chromosome 10q22.3–q24.2 IV locus. Among these, the MMRN2 gene became a candidate gene, as it encodes an extracellular protein multimerin-2 precursor (also known as EMILIN-3, elastin microfibril interface located protein 3 or elastin microfibril interfacer 3) (see http://us.expasy.org/cgi-bin/
niceprot.pl?Q9H8L6). The seven coding exons and exon–intron boundaries of MMRN2 were analyzed for potential mutations in the two probands, but no pathogenic mutation was found. Future systematic analysis of the candidate genes at the 10q22.3–q24.2 locus may identify a new disease gene for IV.

In summary, we identified a previously unreported locus for IV on chromosome 10q22.3–q24.2 in two Chinese families. This genetic locus is the second locus identified for IV. As only one IV gene is identified to date, identification of new genes for IV will help elucidation of the molecular mechanisms for the pathogenesis of IV. Our results provide a framework to identify a previously unreported gene for IV.

Materials and Methods

Patients and genomic DNA

Two Chinese families with IV were identified and characterized at the Affiliated Union Hospital of Huazhong University of Science and Technology. The involved family members received detailed medical examinations by expert dermatologists.

The study complied with the Declaration of Helsinki Principles and was approved by the ethics committee of Huazhong University of Science and Technology (Wuhan, China). After informed consent was obtained, venous blood samples were obtained from affected and unaffected family members and used for isolation of genomic DNA. Genomic DNA was extracted according to standard procedures using the Promega Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI). The DNA was quantified by spectrophotometric analysis and diluted to a concentration of 25 ng l^{- 1} for PCR amplification. The IgE level was measured with a standard assay by the Central Clinical Diagnostic Laboratory at the Affiliated Union Hospital of Huazhong University of Science and Technology.

Genotyping and linkage analysis

Genome-wide linkage screening of the large IV family was performed with 382 polymorphic markers that are spaced every 10 cM on average on chromosomes 1–22 (Linkage Mapping Set MD-10, Applied Biosystems Inc., Foster City, CA). In the fine mapping phase, additional fluorescently labeled primers were selected from the Marshfield Clinic Medical Genetics database (http://research.marshfieldclinic.org/genetics/
GeneticResearch/compMaps.asp).

Multiplex PCR was carried out in a 5 l reaction volume containing 25 ng of genomic DNA, 1 PCR buffer, 0.3 mM dNTP, 1.5 mM MgCl₂, 0.1 M of each primer, and 0.3 U of Taq polymerase (Takara Biotechnology Co., Dalian, China). PCRs were performed in an ABI GeneAmp 9700 PCR System as previously described (Wang et al., 1995, 1996). The PCR product was loaded onto an ABI PRISM 3100 Genetic Analyzer. Genotypes were analyzed using the GeneMapper version 3.0 software (Applied Biosystems Inc.).

Two-point linkage analysis was calculated with the MLINK program of the Linkage 5.2 package (Lathrop and Lalouel, 1984). The inheritance pattern of the disease in the families was assumed to be autosomal dominant. Other assumptions included a penetrance rate of 95% , a phenocopy rate of 0% , a disease allele frequency of 0.0001, and equal recombination fractions () in men and women. The most likely haplotype was constructed by the CYRILLIC version 2.1.5.1 (Cherwell Scientific Publishing Ltd, Reading, UK) software in combination with manual analysis.

Mutational analysis

Candidate genes were identified using the UCSC Genome Browser (http://genome.ucsc.edu/). For the candidate gene, we designed primer pairs that amplify all exons and exon–intron boundaries and used them for PCR amplification. PCR products were gel-purified using the Gel Extraction Mini Kit (Watson Biotechnologies Inc., Shanghai, China) and subjected to DNA sequencing analysis using both forward and reverse primers using the ABI BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems Inc.).

Conflict of Interest

The authors state no conflict of interest.

References

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Acknowledgments

We thank the patients and families for their enthusiastic participation. Without their support, this study would not have been possible. This study was supported by the Chinese Ministry of Science and Technology 863 Project Grant nos. 2002BA711A07 (Q.K.W.), 2006AA02Z476 (Q.K.W.), and the China Natural Science Foundation Grant 30670736 (J.Y.L.). Q.K. Wang is an established investigator of the American Heart Association (0440157N).

SUPPLEMENTARY MATERIAL

Figure S1. Results of genome-wide linkage analysis in Chinese family 1 with IV for markers on chromosomes 1–22.