An Autosomal Recessive Form of Monilethrix Is Caused by ... - Core

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An Autosomal Recessive Form of Monilethrix Is Caused by Mutations in DSG4: Clinical Overlap with Localized Autosomal Recessive Hypotrichosis Abraham Zlotogorski1, Dina Marek2,3, Liran Horev1, Almogit Abu2,3, Dan Ben-Amitai4, Liora Gerad2, Arieh Ingber1, Moshe Frydman2,5, Haike Reznik-Wolf2, Daniel A. Vardy6 and Elon Pras2,5 Monilethrix is a structural defect of the hair shaft usually inherited in an autosomal dominant fashion and caused by mutations in the hHb1, hHb3, and hHb6 keratin genes. Autosomal recessive inheritance in this disease has been sporadically reported. We encountered 12 Jewish families from Iraq, Iran, and Morocco with microscopic findings of monilethrix, but with no evidence of vertical transmission. Since no mutations were found in these three hair keratin genes, we examined nine chromosomal regions containing gene clusters encoding skin and hair genes. On chromosome 18q, a common haplotype in the homozygous state was found among all seven Iraqi patients, but not in 20 controls (Po0.0001). Sequencing of the main candidate gene from this region revealed four different mutations in desmoglein 4 (DSG4). Mutations in DSG4 have been previously reported in localized autosomal recessive hypotrichosis, a disorder that shares the clinical features of monilethrix but lacks the characteristic microscopic appearance of the hair shaft. Our findings have important implications for genetic counseling to monilethrix patients and families, and suggest that DSG4-associated hair disorders may be more common than previously thought. Journal of Investigative Dermatology (2006) 126, 1292–1296. doi:10.1038/sj.jid.5700251; published online 30 March 2006

INTRODUCTION Monilethrix (Mendelian inheritance in man 158000) is a congenital defect of the hair that is inherited as a fully penetrant condition with variable expression (Horev et al., 2000). Affected individuals have normal hair at birth, but within the first few months of life develop fragile, brittle hair that tends to fracture and produce varying degrees of dystrophic alopecia. In the mildest forms only the occipital regions of the scalp are involved; however in severe forms the secondary sexual hair, the eyebrows, and the eyelashes may also be involved. Follicular hyperkeratosis with predilection to the scalp, nape of the neck, and extensor surfaces of the upper arm and thigh is also a characteristic finding in these patients. Light microscopic examination is diagnostic and reveals elliptical nodes of normal thickness and intermittent constrictions, internodes at which the hair easily breaks. In 1

Department of Dermatology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel; 2Danek Gartner Institute of Human Genetics, Sheba Medical Center, Tel Hashomer, Israel; 3Bar Ilan University, Ramat Gan, Israel; 4 Pediatric Dermatology Unit, Schneider Children’s Medical Center of Israel, Petah Tiqva, Israel; 5Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel and 6Dermatology Service, Clalit Health Services, Southern District, Beer-Sheva, Israel Correspondence: Dr Abraham Zlotogorski, Department of Dermatology, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel. E-mail: [email protected]

general, there is a tendency for spontaneous improvement with time especially during puberty and pregnancy, but the condition never disappears completely. Classically, monilethrix is inherited in an autosomal dominant fashion and has been associated with mutations in three keratin genes located on chromosome 12q13: hHb1, hHb3, and hHb6 (Winter et al., 1997a, b; Korge et al., 1999; Horev et al., 2000; van Steensel et al., 2005). However, not all monilethrix patients harbor mutations in these genes and further heterogeneity has been suggested in the past (Richard et al., 1996; Horev et al., 2003). Furthermore, sporadic reports about patients with apparently no family history have raised the possibility that in some cases monilethrix is inherited as an autosomal recessive disease (Hanhart, 1955; Salamon and Schnyder, 1962). We have ascertained 12 Jewish monilethrix families, most from Iraq, but also from Iranian and Moroccan descent, with no evidence of vertical transmission. Sequencing of the three keratin genes, hHb1, hHb3, and hHb6, did not show any mutations in these patients, and therefore we embarked on a candidate gene approach to find the underlying molecular basis of this disorder. In this study, we report the clinical and mutational analysis of 12 families with autosomal recessive monilethrix. RESULTS

Abbreviation: DSG4/DSG4, desmoglein 4 gene/protein

Families and patients

Received 17 September 2005; revised 24 January 2006; accepted 27 January 2006; published online 30 March 2006

In all, 12 family pedigrees are shown in Figure 1. In two of the families (3 and 4) the parents were first cousins. The ethnic

1292 Journal of Investigative Dermatology (2006), Volume 126

& 2006 The Society for Investigative Dermatology

A Zlotogorski et al. DSG4 Mutations in Autosomal Recessive Monilethrix

a

Family 1

Family 5

Family 2

Family 6

Family 9

Family 3

Family 7

Family 10

Family 11

b

Family 4

Family 8

Family 12

c

d

Figure 1. Pedigrees of the 12 monilethrix families.

Table 1. Ethnic origin and mutation results of the 12 autosomal recessive monilethrix families Family no.

Parent 1

Parent 2

Mutation 1

Mutation 2

1–6

Iraq

7, 12

Iraq

Iraq

P267R

P267R

Morocco

P267R

R289X

8–9

Iraq

Iran

P267R

216+1G-T

10

Iran

Iran

763delT

216+1G-T

11

Iraq

Iran

P267R

763delT

Carrier frequency was determined in 100 ethnically matched controls. The carrier’s rate for Iraqi mutation, P267R, for the Iranian mutation, 763delT, and for the Moroccan mutation, R289X, were 1:25, 1:100, and 1:100, accordingly, and absent for the second Iranian mutation, 216+1G-T.

origin of the parents is provided in Table 1. All patients presented with early-onset generalized scalp alopecia within the first days of life with ‘‘black dots’’ appearance. Follicular scalp hyperkeratotic papules with erythema, scaling, dryness, and accompanying pruritus were also noted at infancy. Toward the end of the second year a variable degree of improvement in hair growth was documented, leading to patchy or widespread scalp alopecia (Figure 2a and b). In all patients the posterior scalp was more severely involved (Figure 2a and b). Hairs in general were short and brittle, but sometimes reached a length of up to 10–20 cm. Two or three hair follicles infrequently converged in a single ostium. Eyebrows and eyelashes were sparse, with no body hair, decreased axillary hair, and minimal involvement of pubic hair. The hyperkeratotic papules were most common on the lower posterior scalp and nape of neck (Figure 2a and b), and variable number of papules on the extensor surfaces of the upper arm and thigh and periumbilical region (Figure 2c). Nails, teeth, and sweating were normal. The common hair abnormality was the elliptical nodes, typical of monilethrix (Figure 2d). Other occasional hair abnormalities included pili torti, trichoschisis, trichorrhexis nodosa-like defects, and tapered hairs. Candidate gene studies

In six of the families both parents were of Iraqi origin. We assumed that in these families there is a founder mutation and

Figure 2. Clinical picture of monilethrix. (a, b) Dystrophic alopecia. Note the more severe involvement of the posterior and lower scalp. (a) Scalp (family 3) and (b) neck (family 6) papules. (c) Periumbilical papules (family 1). (d) Typical moniliform hair with periodic widening and narrowing (family 1).

hence these patients would be homozygotes for the same mutation. In such a case all or most patients should show homozygosity for polymorphic markers very close to the disease gene. Since many of the genes that encode components of the skin and hair appear in clusters, we used a linkage disequilibrium candidate gene approach to study nine candidate gene clusters, including the type II keratin cluster on chromosome 12q. We screened the corresponding genomic regions for the presence of microsatellite repeats, and initially used one marker from each cluster to genotype 7 Iraqi monilethrix patients and 20 Iraqi control subjects in search for an allele over-represented in the patients relative to the controls. Results obtained with the marker from clone AC012417 (chromosome 18q) showed that all patients were homozygous for the 219 bp allele, while only three of the controls were heterozygous for the same allele (Po0.001). We therefore identified two additional microsatellite repeats from this cluster, from clones AC079096 and AC009717, and genotyped the patients and the controls. The haplotype comprised of the 216, 219, and 234 bp alleles for markers from clones AC009717, AC012417, and AC079096, respectively (Table S1), and was found in the homozygous state in all of the patients but in none of 20 controls (Po0.0001). Candidate gene analysis. The chromosome 18q gene cluster contains three desmocollin and four desmoglein genes. Two of the desmogleins, designated dsg3 in the mouse, desmoglein 4 (DSG4) in humans, and dsg4 in the mouse and rat, have been previously associated with hair disorders (Montagutelli et al., 1997; Bazzi et al., 2005). Complete sequencing of the whole coding region and the intron boundaries of DSG4 revealed a C-to-G transversion at nucleotide position 799 (Figure 3). This substitution results in a proline-to-arginine change in the protein (P267R) and creates an AciI restriction site in the carrier chromosomes. www.jidonline.org 1293


a T

280 A A

T

T

T

C

C

G

C A

290 C C

T

T

A

T

280 A A

T

T

C

T

C

A

A

140 T G

C

A

T

A

C

G

A

T

T

150 A C

A

A

A

C

140 G

T

A

A

T

T

T

G

C

C

A

A

210 A G

T

A

A

G

T

G

A

T

T

G

C

C

A

A

A

C

G

210 N

C

Wild-type

T

d

G

T

G

T

G

A

G

A

T

A

T

T

A

150 C A

T

A

G

A

A

T

C

Wild-type

250 G

T

G

T

G

A

C

A

A

G

250 G

N

T

G

A

T

216+1G 250 C T

T

C T R289X

Wild-type

c

290 C C

G C P267R

Wild-type

b

C A

G

N

T G A

A

N

N

763delT

Figure 3. Mutations in the DSG4 gene. DNA sequences are shown from unrelated, unaffected control individuals (wild type), and the probands (homozygous mutation P267R and heterozygous mutations; 216 þ 1G-T, 763delT, and R289X). The arrows indicate the site of the mutations.

Using this restriction site, we confirmed the mutation in all seven Iraqi Jewish patients in the homozygous state, and in four out of 100 Iraqi Jewish controls in the heterozygous state, establishing a carrier rate of 1:25. The mutation was not found in 100 controls from other ethnic groups. A database search revealed that the DSG4 has been sequenced in four different species: Mus musculus, Canis familiaris, Bos taurus, and Rattus norvegicus (Jahoda et al., 2004), all of which show a conserved proline at this position. Subsequently, three additional mutations were detected: 763delT and 216 þ 1G-T in patients of Iranian descent and R289X in patients of Moroccan descent (Figure 3 and Table 1). Restriction digestion was used to confirm the mutations and assess the carrier rates (Table S2). 1294 Journal of Investigative Dermatology (2006), Volume 126

DISCUSSION Mutations in DSG4 have been previously described in patients with localized autosomal recessive hypotrichosis, a rare disorder that is characterized by fragile, brittle short hair and follicular hyperkeratosis (Kljuic et al., 2003; Rafiq et al., 2004), but without the microscopic hair shaft changes of monilethrix. A large deletion in DSG4, spanning four exons, has been described in seven Pakistani families (Kljuic et al., 2003; Moss et al., 2004; Rafiq et al., 2004). Lack of the classical hair shaft beading in the previously reported localized autosomal recessive hypotrichosis patients may be explained by the different type of mutations, or by a different genetic background. Alternatively, in some patients the hair beading may be very scarce and overlooked. In some of our


patients, monilethric hairs were found only by using a dermatoscope, which directed the examiners in plucking the distorted monilethric short hairs. Despite the lack of microscopic hair shaft findings, it seems as though there is a considerable clinical overlap between localized autosomal recessive hypotrichosis and monilethrix. Interestingly, mutations in the mouse and rat Dsg4 homologs cause the lanceolate hair phenotype, characterized by sparse, fragile, broken hair shafts, ichtyosiform dermatitis, and follicular dystrophy. Lanceolate hairs, and various other hair abnormalities, raising the possibility of Netherton’s syndrome and periodic nodules that resemble monilethrix were reported in these mice (Sundberg et al., 2000). Structurally, DSG4, as other cadherins, contains a number of domains: a signal sequence; a propeptide of around 130 residues; a single transmembrane domain; an extracellular domain comprised of five tandemly repeat elements; and an N-terminal cytoplasmic domain (Yap et al., 1997). All of the four mutations that we have found occur at the extracellular domain. DSG4 is expressed in the epithelial layers of the hair follicle, where it constitutes part of the desmosome. The desmosome has an important role in cell-to-cell adhesion and signaling, and is crucial for proper hair follicle development and differentiation. Similar to other keratin intermediate filaments, it is presumed that the hair keratins hHb1, hHb3, and hHb6 are structurally bound to the desmosome and consequently influenced by its function. Therefore, it is not surprising that mutations in both components can give rise to a similar phenotype. Mutations in the two other protein families that make up the desmosome, plakin and armadillo, have also been described in disorders involving the skin and the hair follicle (McGrath and Wessagowit, 2005). In retrospect, the clinical picture of autosomal recessive monilethrix is more severe than the dominant one, with more extensive alopecia of the scalp, body, and limb alopecia, and papular rash, which includes the extremities and periumbilical region, features very similar to those that we have previously reported in patients homozygous for a mutation in hHb6 (Zlotogorski et al., 1998). Yet, these clinical differences cannot be used for distinguishing between patients with keratin or DSG4 mutations. Further complicating the picture is the fact that de-novo mutations in the keratin genes have been described in monilethrix patients without a family history (Horev et al., 2003). Thus, such patients may suffer from mutations in one of the keratin genes or in DSG4. MATERIALS AND METHODS The families were recruited at the Hadassah and Sheba Medical Centers. The Institutional Review Board at each center approved the study and participants gave informed consent. The study was conducted according to The Declaration of Helsinki Principles. Overall, DNA samples were analyzed from 24 family members, 15 patients, and nine unaffected individuals. DNA was extracted using a commercial kit (Gentra System Inc., Minneapolis, MN) and primers were designed using the Primer3 software. Amplification of the polymorphic markers was carried out in a 25-ml reaction containing 50 ng of DNA, 13.4 ng of each unlabeled primer, 1.5 mM deoxynucleoside triphosphates, 0.08 mg labeled primer in 1.5 mM MgCl2,

and PCR buffer, with 1.2 U of Taq polymerase (Bio-Line, London). After an initial denaturation of 5 minutes at 951, 30 cycles were performed (941C for 30 seconds, 56–581C for 45 seconds, and 721 for 30 seconds), followed by a final extension of 7 minutes at 721C. For sequencing, the whole coding region and the exon–intron boundaries of DSG4 were amplified and sequenced (Kljuic et al., 2003) using an automated ABI Prism 310 Genetic Analyzer (Perkin-Elmer). Mutations were confirmed and carrier rates were established using restriction assays described in Table S2. CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS We thank the members of the families for their participation in this study. This work was supported in part by the Authority for Research and Development, Hebrew University of Jerusalem (A.Z.). WEB RESOURCES The URLs for data presented herein are as follows: Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/ OMIM/ (for monilethrix and hypotrichosis localized autosomal recessive) Ensembl http://www.ensembl.org/ Santa Cruz (UCSC), http://genome.ucsc.edu/goldenPath/hgTracks.html Single Nucleotide Polymorphism (SNP), http://www.ncbi.nlm.nih.gov/SNP National Center for Biotechnology Information (NCBI), http://www.ncbi.nlm. nih.gov/ Primer 3, http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi

SUPPLEMENTARY MATERIAL Table S1. Polymorphic markers from nine skin and hair gene clusters. Table S2. DSG4 carrier rates in ethnically matched controls.

REFERENCES Bazzi H, Martinez-Mir A, Kljuic A, Christiano AM (2005) Desmoglein 4 mutations underlie localized autosomal recessive hypotrichosis in humans, mice, and rats. J Investig Dermatol Symp Proc 10:222–4 Hanhart E (1955) Erstmaliger Hinweis auf das Vorkommen eines monohybridrezessiven Erbgangs bei Monilethrix (Moniletrichosis). Arch Klaus Stift Vererbungsforsch 30:1–11 Horev L, Djabali K, Green J, Sinclair R, Martinez-Mir A, Ingber A et al. (2003) De novo mutations in monilethrix. Exp Dermatol 12:882–5 Horev L, Glaser B, Metzker A, Ben-Amitai D, Vardy D, Zlotogorski A (2000) Monilethrix: mutational hotspot in the helix termination motif of the human hair basic keratin 6. Hum Hered 50:325–30 Jahoda CA, Kljuic A, O’Shaughnessy R, Crossley N, Whitehouse CJ, Robinson M et al. (2004) The lanceolate hair rat phenotype results from a missense mutation in a calcium coordinating site of the desmoglein 4 gene. Genomics 83:747–56 Kljuic A, Bazzi H, Sundberg JP, Martinez-Mir A, O’Shaughnessy R, Mahoney MG et al. (2003) Desmoglein 4 in hair follicle differentiation and epidermal adhesion: evidence from inherited hypotrichosis and acquired pemphigus vulgaris. Cell 113:249–60 Korge BP, Hamm H, Jury CS, Traupe H, Irvine AD, Healy E et al. (1999) Identification of novel mutations in basic hair keratins hHb1 and hHb6 in monilethrix: implications for protein structure and clinical phenotype. J Invest Dermatol 113:607–12 McGrath JA, Wessagowit V (2005) Human hair abnormalities resulting from inherited desmosome gene mutations. Keio J Med 54:72–9 Montagutelli X, Lalouette A, Boulouis HJ, Guenet JL, Sundberg JP (1997) Vesicle formation and follicular root sheath separation in mice homozygous for deleterious alleles at the balding (bal) locus. J Invest Dermatol 109:324–8 Moss C, Martinez-Mir A, Lam H, Tadin-Strapps M, Kljuic A, Christiano AM (2004) A recurrent intragenic deletion in the desmoglein 4 gene

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underlies localized autosomal recessive hypotrichosis. J Invest Dermatol 123:607–10 Rafiq MA, Ansar M, Mahmood S, Haque S, Faiyaz-ul-Haque M, Leal SM et al. (2004) A recurrent intragenic deletion mutation in DSG4 gene in three Pakistani families with autosomal recessive hypotrichosis. J Invest Dermatol 123:247–8 Richard G, Itin P, Lin JP, Bon A, Bale SJ (1996) Evidence for genetic heterogeneity in monilethrix. J Invest Dermatol 107:812–4 Salamon T, Schnyder UW (1962) Ueber die Monilethrix. Arch Klin Exp Dermatol 215:105–36 van Steensel MA, Steijlen PM, Bladergroen RS, Vermeer M, van Geel M (2005) A missense mutation in the type II hair keratin hHb3 is associated with monilethrix. J Med Genet 42:e19

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Sundberg JP, Boggess D, Bascom C, Limberg BJ, Shultz LD, Sundberg BA et al. (2000) Lanceolate hair-J (lahJ): a mouse model for human hair disorders. Exp Dermatol 9:206–18 Winter H, Rogers MA, Gebhardt M, Wollina U, Boxall L, Chitayat D et al. (1997a) A new mutation in the type II hair cortex keratin hHb1 involved in the inherited hair disorder monilethrix. Hum Genet 101:165–9 Winter H, Rogers MA, Langbein L, Stevens HP, Leigh IM, Labreze C et al. (1997b) Mutations in the hair cortex keratin hHb6 cause the inherited hair disease monilethrix. Nat Genet 16:372–4 Yap AS, Brieher WM, Gumbiner BM (1997) Molecular and functional analysis of cadherin-based adherens junctions. Annu Rev Cell Dev Biol 13:119–46 Zlotogorski A, Horev L, Glaser B (1998) Monilethrix: a keratin hHb6 mutation is co-dominant with variable expression. Exp Dermatol 7:268–72