Arch Dermatol Res (2010) 302:701–703 DOI 10.1007/s00403-010-1035-6
S H O R T CO M MU N I C A T I O N
A novel splice-acceptor site mutation in CDH3 gene in a consanguineous family exhibiting hypotrichosis with juvenile macular dystrophy Syed Kamran-ul-Hassan Naqvi · Zahid Azeem · Ghazanfar Ali · Wasim Ahmad
Received: 24 January 2010 / Revised: 26 January 2010 / Accepted: 27 January 2010 / Published online: 7 February 2010 © Springer-Verlag 2010
Abstract Mutations in CDH3 gene, encoding P-cadherin, are responsible for hypotrichosis with juvenile macular dystrophy (HJMD), which is a rare autosomal recessive disorder. The HJMD is characterized by congenital sparse hair on scalp and progressive severe degenerative changes of the retinal macula which leads to variable degrees of blindness. The present study reports a large consanguineous Pakistani family with six individuals aVected with HJMD. Genotyping using polymorphic microsatellite markers showed linkage of the family to CDH3 gene on chromosome 16q22.1. Sequence analysis of the CDH3 gene revealed a novel splice site mutation (c.IVS10–1 G ! A) in intron 10, which leads to skipping of exon 11 and probably synthesizing a non-functional premature truncated protein. Keywords
HJMD · CDH3 gene · Splice site mutation
Introduction Hypotrichosis with juvenile macular dystrophy (HJMD, MIM 601553), an autosomal recessive human genetic disorder, is characterized by hair loss followed by progressive degeneration of the central retina leading to blindness. Indelman et al. [5] have reported signiWcant diVerences in hair morphology, associated skin abnormalities, and age of onset and severity of visual disability among the HJMD patients. The HJMD syndrome is caused by mutations in P-cadherin (CDH3) gene located on human chromosome 16q22.1 [4, 10]. Recently, mutations in the CDH3 gene
were also reported to cause ectodermal dysplasia, ectrodactyly and macular dystrophy (EEM) syndrome (MIM 225280) [7]. Patients of both these diseases exhibit sparse hair and macular dystrophy of the retina, however, patients with EEM syndrome also show the additional features of split hand/foot malformations (SHFM) [7]. The P-cadherin encoding CDH3 gene contains 16 exons spanning 55 kb on human chromosome 16q22.1. It is a member of classical cadherin family that forms the transmembrane core of adherens junctions. Several studies have reported the involvement of classical cadherins including E-cadherin and P-cadherin, encoded by CDH1 and CDH3 genes, respectively, in biological processes such as cell signaling, cell recognition, hair follicle morphogenesis and limb development [9]. The CDH3 expresses in wide variety of tissues including retinal pigmented epithelium, hair follicle and limb bud [9]. The P-cadherin protein is regulated by a transcription factor p63 [9], which is a key regulator of broader cell adhesion gene expression programs [2]. Interestingly mutations in p63 gene also result in syndromic and non-syndromic SHFM [3]. Similarities in phenotypes of patients with mutations in CDH3 and p63 genes predict that interaction between these two genes playing important role in the development of hair follicle and limb bud development [9]. In the present study, we report our second large Pakistani family with autosomal recessive HJMD. DNA sequence analysis revealed a novel splice site mutation.
Patients and methods S. Kamran-ul-Hassan Naqvi · Z. Azeem · G. Ali · W. Ahmad (&) Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan e-mail:
[email protected]
In the present study, a consanguineous Sindhi-speaking Pakistani family with a rare autosomal recessive HJMD has been ascertained from a remote region of Pakistan (Fig 1).
123
702
Arch Dermatol Res (2010) 302:701–703
Fig. 1 a Pedigree of family with autosomal recessive hypotrichosis with juvenile macular dystrophy (HJMD). AVected individuals are represented by Wlled symbols while clear symbols are for unaVected family members. The diseaseassociated haplotypes are shown beneath each genotyped individual. b Clinical presentation of an aVected individual (III-1) with HJMD phenotype. Note sparse hairs on the scalp
Approval of the study was provided by the Quaid-i-Azam University Institutional Review Board (IRB). Informed consent was obtained from all those members of the family who participated in the study. The aVected individuals of the present family were born with sparse scalp hair that had limited growth throughout life. In two aVected females (IV-2, IV-3), scalp hairs were completely missing at the time of birth. In all the aVected individuals there was thin and sparse growth of eyebrows and eyelashes, pubic and pelvic hair. Skin, Wngernails, toenails and teeth showed normal growth (Fig. 1). The aVected individuals reported progressive loss and weakness of the eyesight at the age of 12–20 years. Full-Weld eletroretinogram (ERG)-based electrophysiological test was performed on two patients (II-3, III-1) of the present family. Fundus examination revealed pigmentary abnormalities at the posterior pole in both the patients. The ERG deWcit was similar for light and dark adapted responses. The fundus picture and ERG responses were consistent with macular disorder in the patients. Blood samples were collected from six aVected (II-2, II-3, III-1, III-2, IV-2, IV-3) and two unaVected (IV-1, IV-4) individuals of the family. Genomic DNA was extracted following a standard protocol and diluted to 40 ng/ul for ampliWcation by polymerase chain reaction (PCR). The ampliWcation of microsatellite markers was performed according to a standard protocol. PCR products were resolved on 8% non-denaturing polyacrylamide gel, stained with ethidium bromide and genotypes were assigned by visual inspection.
123
To search for an underlying mutation in CDH3 gene, all of its 16 exons and splice junctions sites were PCR ampliWed from genomic DNA of all the 6 aVected and 2 normal individuals of the family using primers designed from intronic sequences of the gene. The ampliWed PCR products were puriWed using the Rapid PCR PuriWcation System (Marligen Biosciences, Ijamsville, MD, USA) and sequenced in an ABI Prism 310 automated DNA sequencer, using Big Dye Terminator Cycle Sequencing Kit (PE Applied Biosystems USA). Sequence variants were identiWed via BIOEDIT sequence alignment editor version 6.0.7. One hundred and Wfty normal control individuals were screened for the potential sequence variant found in the present study.
Results Considering clinical features of aVected individuals of the family, it was tested for linkage to CDH3 gene on chromosome 16q22.1. The microsatellite markers (D16S3393, D16S3050, D16S3095, D16S2624, D16S3033) used in genotyping were fully informative and all six aVected individuals were homozygous with these markers, suggesting linkage of the family to CDH3 gene. Sequence analysis of the CDH3 gene in the aVected individuals revealed a novel splice site variant in intron 10 (c.IVS10–1 G ! A). The obligate carriers carry this mutation in heterozygous state (Fig. 2). To ensure that the mutation does not represent a neutral polymorphism in this
Arch Dermatol Res (2010) 302:701–703
703
to play a role in binding of P-cadherin to Ca2+ and determination of the P-cadherin folding conformation. The folding conformation in turn inXuences cell–cell adhesion activity [1]. The splice site mutation (c.IVS10–1 G ! A), reported here, probably disturbs the mechanisms of cell–cell adhesion which are important for normal development of hair follicle, retinal pigment and ectodermal structures. Acknowledgments We are thankful to all the members of the family, for their enthusiastic participation and co-operation for the present case study. The work presented here was funded by Higher Education Commission (HEC), Islamabad, Pakistan. Syed Kamran-ul-Hassan Naqvi was supported by indigenous PhD fellowship from HEC, Islamabad, Pakistan.
References
Fig. 2 Automated DNA sequence analysis of CDH3 gene mutation: a a homozygous aVected individual (II-3), b a heterozygous carrier individual (IV-1), c a control individual. A single base pair G in control individual c is substituted into A in the aVected individual a. Arrows indicate position of substitution of G nucleotide
population, a panel of 150 unrelated unaVected ethnically matched control individuals was screened for the mutation and it was not identiWed outside the family.
Discussion To date, 13 mutations including 3 deletions, 1 insertion, 2 missense, 1 nonsense, 2 compound heterozygous and 2 splice site have been reported in the CDH3 gene in patients with HJMD [6, 9]. The mutation (c.IVS10–1 G ! A), reported in the present study, is the third splice site mutation identiWed in the CDH3 gene and is predicted to cause out of frame skipping of exon 11 and generates a pre-termination codon in exon-12. As a result, it is possible that this mutation generates a truncated protein lacking most of the essential domains of CDH3 protein. Alternatively, CDH3 mRNA containing a pre-termination codon might be degraded due to nonsense-mediated decay [8]. The CDH3 protein contains Wve extracellular domains (EC1–5), a transmembrane region and a short intracellular tail. The EC4 domain encoded by DNA sequence of exon11 contains a DRE sequence motif, which has been shown
1. Boggon TJ, Murray J, Chappuis-Flament S, Wong E, Gumbiner BM, Shapiro L (2002) C-cadherin ectodomain structure and implications for cell adhesion mechanisms. Science 296:1308–1313 2. Carroll DK, Carroll JS, Leong CO, Cheng F, Brown M, Mills AA, Brugge JS, Ellisen LW (2006) p63 regulates an adhesion programme and cell survival in epithelial cells. Nat Cell Biol 8:551–561 3. Ianakiev P, Kilpatrick MW, Toudjarska I, Basel D, Beighton P, Tsipouras P (2000) Split-hand/split-foot malformation is caused by mutations in the p63 gene on 3q27. Am J Hum Genet 67:59–66 4. Indelman M, Eason J, Hummel M, Loza O, Suri M, Leys MJ, Bayne M, Schwartz FL, Sprecher E (2007) Novel CDH3 mutations in hypotrichosis with juvenile macular dystrophy. Clin Exp Dermatol 32:191–196 5. Indelman M, Hamel CP, Bergman R, Nischal KK, Thompson D, Surget MO, Ramon M, Ganthos H, Miller B, Richard G, Lurie R, Leibu R, Russell-Eggitt I, Sprecher E (2003) Phenotypic diversity and mutation spectrum in hypotrichosis with juvenile macular dystrophy. J Invest Dermatol 121:1217–1220 6. Jelani M, Chishti MS, Ahmad W (2009) A novel splice-site mutation in the CDH3 gene in hypotrichosis with juvenile macular dystrophy. Clin Exp Dermatol 34:68–73 7. Kjaer KW, Hansen L, Schwabe GC, Marques-de-Faria AP, Eiberg H, Mundlos S, Tommerup N, Rosenberg T (2005) Distinct CDH3 mutations cause ectodermal dysplasia, ectrodactyly, macular dystrophy (EEM syndrome). J Med Genet 42:292–298 8. Maquat LE (1996) Defects in RNA splicing and the consequence of shortened translational reading frames. Am J Hum Genet 59:279–286 9. Shimomura Y, Wajid M, Shapiro L, Christiano AM (2008) P-cadherin is a p63 target gene with a crucial role in the developing human limb bud and hair follicle. Development 135:743–753 10. Sprecher E, Bergman R, Richard G, Lurie R, Shalev S, Petronius D, Shalata A, Anbinder Y, Leibu R, Perlman I, Cohen N, Szargel R (2001) Hypotrichosis with juvenile macular dystrophy is caused by a mutation in CDH3, encoding P-cadherin. Nat Genet 29:134–136
123
