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Two Different in the Same Codon of a Type II (hHb6) in Patients with

Eve G. Pearce, S. Kaye Smith, Sean W. Lanigan,* and Paul E. Bowden Department of Dermatology, University of Wales College of Medicine (UWCM), Heath Park, Cardiff, U.K.; *Princess of Wales Hospital, Coity Road, Bridgend, U.K.

Monilethrix is an autosomal dominant hair disorder whereas in patient B, the transition was G to A characterized by a beaded appearance of the hair due yielding a lysine substitution (GAG to AAG: E402K). to periodicthinning of the shaft. The phenotype The sequence of the 1A helical regions of hHb1 and shows variable penetrance and results in hair fragility hHb6 as well as the 2B helical region of hHb1, were and patchy dystrophic alopecia. Mutations of the normal. Unaffected relatives did not have the hHb6 helix-encoding region in two hair-speci®c and this codon was found to be highly (hHb1 and hHb6) have been identi®ed. We have conserved showing no alteration in the normal now investigated two unrelated monilethrix patients population (100 alleles examined). Both mutations and identi®ed two different novel heterozygous point disrupted a Taq I restriction site and restriction mutations of the same codon in exon 7 of the hHb6 fragment length polymorphism analysis showed that gene. Dystrophichair samples obtained from both a diagnostic 361 bp fragment could con®rm the patients showed the typical beaded appearance by mutation. Thus, two new point mutations of the scanning electron microscopy. Both mutations hair-speci®c keratin gene hHb6 have been identi®ed affected the ®rst base of codon 402 (glutamic acid). in this geneticdisease. Key words: gene mutation/hair In patient A, a G to C transition occurred causing a disease/human/intermediate ®laments. J Invest Dermatol glutamine substitution (GAG to CAG: E402Q) 113:1123±1127, 1999

onilethrix isa rare autosomaldominant hair These hair keratins are obligate heteropolymers, which assemble disorder with variable expression (Cockayne, into an IF network within the hair trichocytes(Heid et al, 1988a, b). 1933) and large family pedigreeshave been Molecular cloning studies have revealed sequence information M described (Alexander and Grant, 1958). Its for more than four type I hair keratins, indicating that this subgroup clinical appearance ischaracterized by brittle, is more extensive than studies had indicated. Published fragile hair, which is prone to fracture close to the scalp, producing sequence information includes: hHa1 (Fink et al, 1995), hHa2 dystrophic alopecia. In mild cases, this gives rise to occipital hair (Rogers et al, 1996), hHa3-I and hHa3-II (Rogers et al, 1995b), loss, whereas total baldness throughout life has been observed in hHa5 (Rogers et al, 1996), hHa4, hHa6, hHa7, and hHa8 (Rogers severe phenotypes. In the most severe cases of the disease, defects of et al, 1998). According to the heteropolymer pair rule, the type II secondary sexual hair, eyebrows, eyelashes, and nails have also been subfamily must be as complex as the type I. Published sequence data described (McKee and Rosen, 1916). for the type II hair-speci®c keratins, however, is limited to hHb1, Hair fragility in monilethrix iscausedby periodic hair thinning hHb6, hHb3, and hHb5 (Rogers et al, 1995a, 1997; Bowden et al, producing a visible beaded appearance of dystrophic . It is the 1998). unique appearance of regular narrow or constricted internodes The causative defect in monilethrix has been linked to the type II between normal thickness hair from which the disease derives its keratin gene cluster at 12q13 (Healy et al, 1995; Stevens et al, 1996). name. Ultrastructural studies of dystrophic hair in monilethrix Genetic linkage information and the observed keratin ®lament patientsidenti®ed that hair shaftthinning wasdue to a degeneration disruption in the internodes of monilethrix hair suggested that a of hair cortical matrix cellsand thusa disruptionto the type II gene mutation might cause the observed keratinization of internodal areasresulted(Ito et al, 1984, 1990). pathogenesis. Human hair-speci®c keratins, members of the intermediate Keratin gene mutations have previously been found to cause ®lament (IF) multigene family, are one of four specialized types of many disorders of the epithelium that are characterized by ®lament structural protein that form the hair ®ber. Studies of hair protein disruption (Corden and McLean, 1996; Rothnagel, 1996; Fuchs extractshave identi®ed eight major hair-speci®ckeratins(Bowden and Cleveland, 1998). Recently, mutationsin one of the type II et al, 1987). These can be divided into type I acidic hair keratins hair-speci®c keratin genes (hHb6) have been reported. Thus, from (Ha1±4) and type II neutral-basic hair-speci®c keratins (Hb1±4). a total of eight monilethrix family pedigreesreported, two different mutations affecting the same hHb6 codon (glutamic acid at position Manuscript received May 4, 1999; revised June 30, 1999; accepted for 410) were found in the original study (Winter et al, 1997b). The publication August 17, 1999. ®rst, a G to A transition in base 1 of the codon, yielded a lysine Reprint requests to: Dr. P. E. Bowden, Department of Dermatology, University of Wales College of Medicine (UWCM), Heath Park, Cardiff, substitution whereas the second, yielded an aspartic acid substitu- CF14 4XN, U.K. Email: [email protected] tion due to a G to T transition in base 3 of the same codon.

0022-202X/99/$ 14.00 ´ Copyright # 1999 by The Society for Investigative Dermatology, Inc. 1123 1124 PEARCE ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 1. Family history, hair morphology, and clinical phenotype of monilethrix. Pedi- greesof monilethrix patientsA (a ) and B (b) show no family history. Affected patients are indicated by solid symbols and unaffected are shown by open symbols.Anasterisk indicatesindividualsfrom whom genomic DNA wasobtained. SEM images from the hair of patient B include: (c) dystrophic beaded hair and (d) hair ®ber internodal breakage. Clinical photograph (e) shows patient B at 12 mo of age indicating widespread alopecia.

Whereasthe mutation wasoriginally designatedE410K, however, family history. The two patients both displayed the typical variable fragile thiswasrenamed E413K according to the complete hHb6 gene brittle hair producing areasof stubbledue to hair ®ber fracture. sequence and amino acid residue numbering therein (Bowden et al, 1998). Since thisoriginal study,Winter et al (1997b) have Scanning electron microscopy (SEM) Piecesof affected hair from both patientsand their normal relativeswere cut, mounted on cylindrical elucidated the equivalent E413K mutation in hHb1, another of metal SEM stubs, and coated with gold. Specimens were loaded, examined the type II hair-speci®c keratin gene family. Also recent studies and photographed using a scanning electron microscope (model Joel 840A). identi®ed a novel E402K mutation in the 2B helix encoding Photographswere taken at variousmagni®cationsbefore developing and domain (Winter et al, 1998) and the novel N114H and N114D printing. mutationsin the 1A helix encoding domain 1 of the hHb6 gene. Investigation of the 1A and 2B helix encoding domains of hHb1 Genomic DNA and polymerase chain reaction (PCR) and hHb6 have identi®ed the association of point mutations in ampli®cation Venousblood wasdrawn from both patientsand one of 2 the unaffected relatives. Genomic DNA from the monilethrix and control these regions with cases of monilethrix (Korge et al, 1998; samples was isolated using a standard DNA extraction kit (Nucleon Zlotogorski et al, 1998). BACC2, Scotlab, Strathclyde, U.K.). DNA encoding the a-helical In thisstudy,we now report two different novel mutationsof subdomains (1A and 2B) of two type II hair keratin genes (hHb1 and codon 402 in the 2B helical domain of hHb6 in two unrelated hHb6) wasampli®ed by PCR. Synthetic oligonucleotide primer pairswere monilethrix cases. Restriction fragment length polymorphism designed from published sequence information (Bowden et al, 1998; (RFLP) analysis with Taq I con®rms the presence of these Rogers et al, 1995a). To amplify the 1A and 2B subdomains primer pairs to mutations. exons 1 and 7 were designed. Primer sequences are detailed as follows: hHb1 (1A helix) forward, 5¢-GTCCTCTCTGCAACCCAAACGT- MATERIALS AND METHODS CCAGG-3¢ and reverse, 5¢-GGAGGCTCAGTCCCTCAC-3¢; hHb1 (2B helix) forward, 5¢-TGGTGGGGAGTGTGGTCTCATTTG-3¢ and Patients Two unrelated cases of monilethrix were studied. The ®rst case reverse, 5¢-ATAGGGTAGGGTCCACAGCTGGTC-3¢; hHb6 (1A helix) was Welsh (patient A) and the second Greek (patient B). Both were forward, 5¢-CATCCTCAGAACCTCCTCTCTTCC-3¢ and reverse, 5¢- suspected cases of a spontaneous mutation causing monilethrix with no CCATCAGACTGGGATCTCAGGCAG-3¢ and hHb6 (2B helix) forward, 5¢-TGGTGGGGAGCATGGTCTCATCGA-3¢ and reverse, 5¢- 1Korge BP, Hamm H, Traupe H, et al: Point mutationsin the 1A ATGCTGCCAGGAGTGTGAGGACAG-3¢. domain (N114) of the hair keratin hHb6 in monilethrix patients: A new PCR reactionswere carried out in 20 ml volumesusing1 3 PCR buffer hotspot for mutations? J Invest Dermatol 112:638, 1999a (abstr.) containing: 50 mM Tris±HCl pH 8, 100 mM NaCl, 0.1 mM ethylenedia- 2Korge BP, Hamm H, Traupe H, et al: Recurrent point mutationsin mine tetraacetic acid, 1 mM dithiothreitol, 50% glycerol and 1% Triton X- human type II hair keratinshHb1 and hHb6: A frequent causeof 100 with 2 mM ddNTPs, and 120 ng of each primer. One unit of Taq monilethrix. Arch Dermatol Res 291:122, 1999b (abstr.) Polymerase (Promega, Southampton, U.K.) was added after a hot start VOL. 113, NO. 6 DECEMBER 1999 HAIR KERATIN MUTATIONS IN MONILETHRIX 1125

Figure 3. Con®rmation of hHb6 mutations with Taq I RFLP. RFLP analysis for both the G to C (patient A) and G to A (patient B) point mutationsin exon 7 of the hHb6 gene. PCR ampli®ed regionscontaining these mutations were digested with Taq I prior to analysis on a 1.5% agarose gel (stained with ethidium bromide). Both mutations destroy a Taq I restriction site producing a diagnostic fragment of 361 bp for the mutant allele with the two digested fragments (238 bp and 123 bp) representing the normal allele.

of codons402 and 403 in hHb6 encodesa Taq I restriction site (TCGA). Restriction digestion of PCR products using Taq I (GibcoBRL, Paisley, U.K.) at 65°C for 3 h wasperformed on both monilethrix and control samples. The samples were analyzed by agarose gel electrophoresis, the DNA visualized with ethidium bromide staining and recorded on Polaroid ®lm (type 667). Figure 2. Automated sequencing demonstrates mutations in the hHb6 gene. The sequence data shown represents a portion of exon 7 of RESULTS the hHb6 gene, which encodesthe 2B helix of the hHb6 protein: (a ) normal DNA (GAG: residue E402) (b) affected patient A (CAG: E402Q) Clinical description and hair morphology The two and (c) affected patient B (AAG: E402K). Monilethrix patientsA and B individuals studied with monilethrix, designated patients A and B both have heterozygouspoint mutationsat residue402 (G to C and G to A, were from two unrelated familiesand in both casesthemutations respectively, as indicated by the arrowhead). were spontaneous, showing no family history (Fig 1a, b). The typical beaded appearance of dystrophic hair from both monilethrix procedure (94°C for 7 min). The PCR conditions comprised 35 cycles: 30 s patients was observed with SEM. A constricted internodal 94°C, 50 s61 °C, 3 min 72°C with completion of the PCR reaction being alternating pattern wasapparent in hair samplesfrommonilethrix followed by 15 min at 72°C and cooling to 4°C. PCR productswere patients( Fig 1c). The internodal areasshowedlongitudinal ridges assessed by running an aliquot (4 ml) on a 1.5% agarose gel. DNA was compared with the normal smooth surface of the hair cuticle visualized with ethidium bromide and recorded on Polaroid ®lm (type 667). (Gummer et al, 1981). Typically, monilethrix hair breaksin the internodal region (Fig 1d) and the resulting clinical phenotype is Automated sequencing of PCR products PCR productswere that of partial or complete alopecia asshownfor one of the patients initially precipitated with a polyethylene glycol mix (26.2% polyethylene (Fig 1e). Hair doesgrow aspatientsage but remainsfragile and glycol 8000, 6.6 mM MgCl2, 0.6 M NaOAc pH 5.2) and the pelletswashed breakseasily. with 70% ethanol (Rosenthal et al, 1993). Cycle sequencing was performed using dye terminators (ABI PRISM BigDye Terminator cycle sequencing Identi®cation of novel mutations in the hHb6 gene Direct ready reaction kit from Applied Biosystems, Foster City, CA) in 0.5 ml sequencing of PCR ampli®ed regions of two hair-speci®c keratin tubes. The sequence reactions were combined with the speci®c forward or genesrevealed that exon 1 (1A helix encoding) of both hHb1 and reverse keratin primers (as mentioned above). Thermal cycler conditions hHb6, and exon 7 (2B helix encoding) of hHb1 were normal in (Perkin Elmer Cetus) consisted of 96°C for 30 s, 50°C for 15 sfollowed by both monilethrix patients. Exon 7 sequence analysis of the hHb6 60°C for 4 min for 25 cycles. Analysis of the sequencing reactions was gene, however, revealed two heterozygouspoint mutations performed using electrophoresis on the ABI Prism 377, version 3.0 (Fig 2a±c), affecting the ®rst base of codon 402 (glutamic acid). (Applied Biosystems). The precise alteration differed in each patient, the ®rst mutation RFLP analysis Both monilethrix mutationswere con®rmed by RFLP (patient A) was a G to C transition yielding a glutamine substitution analysis involving restriction enzyme digestion. The normal DNA sequence (GAG to CAG: E402Q) whereasthe secondmutation (patient B) 1126 PEARCE ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY was a G to A transition leading to a substitution of glutamic acid by We thank Mrs Carol Mower for technical assistance with SEM and the Electron lysine (GAG to AAG: E402K). The E402K mutation was not Microscopy unit (UWCM, Cardiff) for use of the Joel microscope. found in an unaffected relative of patient B. Both hHb6 mutations were absent in 10 unrelated, healthy control individuals by direct sequencing. REFERENCES Mutations in hHb6 con®rmed by RFLP analysis The two Alexander J O'D, Grant PW: Monilethrix. Report of three cases with extensive family history. Scot Med J 3:356±360, 1958 new mutations could be con®rmed by RFLP analysis, as the loss of Bowden PE. Keratins and other epidermal . In: Priestly GC (ed.) Molecular a Taq I site was associated with both sequence alterations. This Aspects of Dermatology, Chapter 2. 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