ORIGINAL ARTICLE See related commentary on pg 540

Mutations in the Lipase H Underlie Autosomal Recessive Woolly Hair/Hypotrichosis Yutaka Shimomura1, Muhammad Wajid1, Lynn Petukhova1, Lawrence Shapiro2,3 and Angela M. Christiano1,4

Woolly hair (WH) is characterized by the presence of fine and tightly curled hair. WH can appear as a symptom of some systemic diseases, or without associated findings (nonsyndromic WH). Nonsyndromic WH is known to be inherited as either an autosomal-dominant (OMIM 194300) or recessive (ARWH; OMIM 278150) trait. In this study, we identified 11 consanguineous families of Pakistani origin with ARWH, as well as associated features including sparse and hypopigmented hair shafts. We first checked for mutations in the P2RY5 gene, which encodes an orphan G-protein-coupled receptor that we recently identified as a cause of ARWH. However, none of the 11 families had mutations in the P2RY5 gene. To identify the disease , we performed linkage studies in one of these families using the Affymetrix 10K array, and identified a region of suggestive linkage on 3q27. This region contains the lipase H (LIPH) gene which has been recently shown to underlie an autosomal-recessive form of hypotrichosis. Mutation analysis resulted in the identification of a total of 5 pathogenic mutations in the LIPH of all 11 families analyzed. These results show that LIPH is a second causative gene for ARWH/hypotrichosis, giving rise to a phenotype clinically indistinguishable from P2RY5 mutations. Journal of Investigative Dermatology (2009) 129, 622–628; doi:10.1038/jid.2008.290; published online 2 October 2008

INTRODUCTION hair shaft anomaly characterized by node and internode Human hair follicles (HFs) share a common structural formation, is observed in affected individuals with monile- organization, in which the growth of the hair shaft originates thrix (OMIM 158000) (Stratigos and Baden, 2001), as well as from the matrix region and is encased in the root sheath those with localized autosomal recessive hypotrichosis components composed of several distinct cell layers. How- (OMIM 607903) (Shimomura et al., 2006; Schaffer et al., ever, hair texture shows significant variations among in- 2006; Zlotogorski et al., 2006). Causative for each of dividuals and different populations. In addition, it is known these diseases have been previously identified (Winter et al., that some hereditary diseases show characteristic hair shaft 1997; Chavanas et al., 2000; Kljuic et al., 2003; Weeda et al., anomalies. For example, affected individuals with trichothio- 1997). dystrophy (OMIM 601675) exhibit a so-called ‘‘tiger tail hair’’ Woolly hair (WH) is a group of hair shaft disorders, which which is characterized by alternating light and dark bands is characterized by fine and tightly curled hair (Chien et al., along the hair shaft under polarized microscopy (Stratigos 2006). WH can appear as a part of several syndromes, such and Baden, 2001). The hair shaft of patients with Netherton as Naxos disease (OMIM 601214) and Carvajal syndrome syndrome (OMIM 256500) typically shows trichorrhexis (OMIM 605676), both of which are characterized by invaginata which is also known as bamboo hair (Stratigos cardiomyopathy, palmoplantar keratoderma, and WH, and and Baden, 2001). Furthermore, moniliform hair, which is a are caused by mutations in the plakoglobin (McKoy et al., 2000) and desmoplakin (Norgett et al., 2000) genes, 1Department of Dermatology, College of Physicians and Surgeons, Columbia respectively. In addition to these syndromes, WH without University, New York, New York, USA; 2Department of Biochemistry and associated findings (nonsyndromic WH) has also been Molecular Biophysics, Columbia University, New York, New York, USA; described. Nonsyndromic WH can show either an auto- 3Department of Ophthalmology, Columbia University, New York, New York, USA and 4Department of Genetics and Development, Columbia University, somal-dominant (OMIM 194300) (Hutchinson et al., 1974) New York, New York, USA or recessive (ARWH; OMIM 278150) (Salamon, 1963; Correspondence: Dr Angela M. Christiano, Department of Dermatology, Hutchinson et al., 1974) inheritance pattern, but only a College of Physicians and Surgeons, Columbia University, 630 West 168th limited number of cases have been reported to date. Street VC-1526, New York, New York 10032, USA. We recently studied six consanguineous families of E-mail:[email protected] Pakistani origin with ARWH. Affected individuals showed Abbreviations: WH, woolly hair; HF, hair follicle; PTC, premature termination codon; LPA, lysophosphatidic acid not only WH, but also varying degrees of sparse hair or Received 19 May 2008; revised 3 August 2008; accepted 12 August 2008; hypotrichosis (Shimomura et al., 2008). All families published online 2 October 2008 showed clear linkage to chromosome 13q14.2–14.3, and

622 Journal of Investigative Dermatology (2009), Volume 129 & 2009 The Society for Investigative Dermatology Y Shimomura et al. LIPH Mutations Underlie ARWH

we identified homozygous pathogenic mutations in the RESULTS P2RY5 gene in all six families (Shimomura et al., 2008). Clinical features The P2RY5 gene encodes a heptaspan transmembrane G- Families 1–11 are consanguineous families of Pakistani origin protein-coupled receptor and is a nested gene residing within with various clinical abnormalities in the HF, ranging from intron 17 of the RB1 gene. P2RY5 is expressed abundantly in WH to sparse hair (Figure 1a–d; Figures S1 and S2). Pedigrees both Henle’s and Huxley’s layers of the inner root sheath of of all families were consistent with autosomal recessive the HF (Shimomura et al., 2008). inheritance. Clinical features were present at birth. All In this study, we ascertained 11 consanguineous Pakistani affected individuals in Families 1, 2, 6, 7, and 9 show a families with ARWH, as well as associated features such as distinct WH phenotype (Figures S1 and S2). The hair over the sparse and depigmented hairs. Surprisingly, we did not find entire scalp region is tightly curled, and the hair density is mutations in the P2RY5 gene in any of these families. Linkage variable from normal to less dense. The hair of some affected analysis in one of these families with the Affymetrix 10K array individuals is light-colored as compared with the typical dark led to the identification of a region showing linkage on brown/black hair in this population. Facial and body hairs chromosome 3q27, in the region of the lipase H (LIPH) gene. appear normal. Under light microscopy, plucked hairs of an It was recently reported that a common founder mutation in affected individual of Family 1 show dystrophic features the LIPH gene causes autosomal recessively inherited without obvious root sheath components (Figure S1). The hair hypotrichosis (OMIM 604379) in Russian populations shaft is twisted (Figure S1) and a trichorrhexis nodosa-like (Kazantseva et al., 2006). More recently, a second homozygous anomaly is frequently observed (Figure 1e). In addition, the mutation in LIPH was identified in a Pakistani family affected distal portion displays a tapered end (Figure 1f). These hair with an autosomal recessive hypotrichosis (Ali et al., 2007). shaft anomalies strongly suggest a hair growth defect even The LIPH gene encodes a member of phospholipase A1 though they are not specific for WH. family (Sonoda et al., 2002) and is expressed abundantly in In contrast to these five families, all affected individuals of human HF mRNA as detected by reverse transcriptase–PCR, Families 5, 10, and 11 exhibit primarily sparse scalp hair that although its precise localization within the HF was not is depigmented, and slightly curled (Figure S2). Eyebrow and determined (Kazantseva et al., 2006). Direct sequencing eyelashes, as well as body hairs, are also sparse. Perifollicular analysis of our families resulted in the identification of a total papules are not obvious. In addition, affected individuals of of 5 pathogenic mutations in the LIPH gene of all 11 families Families 3, 4, and 8 show significant differences in the studied. Our findings further define the pathogenesis of severity of the phenotype within a family (Figure 1a–d; ARWH/hypotrichosis in humans. Figures S1 and S2). Some affected individuals show mainly

abc

d e f

Figure 1. Clinical features of Pakistani families with ARWH/hypotrichosis. (a, b) Affected individuals in Family 3. (c, d) Affected individuals in Family 8. Plucked hairs of an affected individual in Family 1 shows a trichorrhexis nodosa-like anomaly (e), and a tapered end (f). Scale bars: 100 mm.

www.jidonline.org 623 Y Shimomura et al. LIPH Mutations Underlie ARWH

WH, whereas others in the same family exhibit primarily Identification of mutations in the LIPH gene sparse hair. The hair growth of affected individuals of all 11 On the basis of the results obtained from linkage analysis, we families is slow, and stops at a few inches. It is noteworthy first performed direct sequencing analysis of the LIPH gene in that most affected individuals with sparse hair originally Family 1, and identified a mutation in LIPH. All affected showed WH at birth, but the sparse hair phenotype became individuals of Family 1 are homozygous for a one nucleotide prominent after the ritual shaving, which is usually performed deletion at position 682 in exon 5 of LIPH, designated a week after birth. Some affected individuals have also 682delT (Figure S3a). We subsequently sequenced the LIPH lost their facial and body hairs with aging. Affected gene in the other 10 families, which led to the identification individuals in all 11 families show normal teeth, nails, and of pathogenic mutations in the LIPH gene in all families. sweating, and do not show palmoplantar hyperkeratosis. Affected individuals in Family 2 are homozygous for a There was no family history of heart disease, cancers or 322T4C transition in exon 2, which results in a substitution neurologic abnormalities. from tryptophan to arginine at amino acid 108 (W108R) (Figure S3b). Affected individuals in Family 3 carry a Linkage to chromosome 3q27 homozygous 624delT mutation in exon 4 of the LIPH gene Using the genomic DNA of affected individuals of all 11 (Figure S3c). Affected individuals in Families 4, 5, and 6 share families, we first performed direct sequencing analysis of the the same homozygous mutation 659-660delTA in exon 5 of P2RY5 gene following the methods described previously the LIPH gene (Figure S3d; Table 1). (Shimomura et al., 2008). None of these families had Finally, the most deleterious mutation was identified in the mutations in the P2RY5 gene (data not shown). We then LIPH gene of Families 7–11. As neither exon 7 nor exon 8 was performed a whole genome scan on Family 1 using the amplified from the DNA of all affected individuals of these Affymetrix 10K SNP array. Autozygosity mapping identified a five families, we designed additional primer pairs at intron 6 region of excess homozygosity shared among affected and intron 8, respectively (E7F2 and E8R2 primers; Figure individuals on chromosome 3q27.1–27.3 with a LOD score S3e; Table S2). PCR with E7F2 and E8R2 primers amplified a of 2.00, suggestive of linkage (Figure 2). Haplotype analysis fragment of 183 bp in size from the DNA of affected and identified three clusters of single-nucleotide polymorphisms carrier individuals, whereas this fragment was not amplified in this region that showed correlated inheritance patterns. from control individuals (Figure S3e). Direct sequencing of Parametric linkage analysis of the haplotypes indicated that the PCR product clearly demonstrated that affected indivi- the maximimum LOD score occurred for the third haplotype, duals in all five families are homozygous for a large deletion, spanning from 187.4 to 189.1 Mb (Z ¼ 1.09). The LIPH gene 5,290 bp in size, including both exons 7 and 8 of LIPH, thus is located at position 186.7 Mb on chromosome 3q27 designated Ex7_8del (Figure S3e). To our knowledge, all five (Figure 2), and given its role in hypotrichosis (Kazantseva mutations identified in this study were not reported pre- et al., 2006; Ali et al., 2007), it became a leading candidate viously. Screening assays excluded the existence of all 5 gene. mutations in 100 unrelated healthy control individuals (200 ) of Pakistani origin (Figure S3a–e and data not shown).

3 Haplotype analysis of founder mutations Autozygosity mapping To determine whether the mutations Ex7_8del and 659- 2.5 Linkage analysis 660delTA are founder mutations in Pakistani populations, we 3q27.1-27.3 performed haplotype analysis using microsatellite markers around and within LIPH gene. All affected individuals in 2 Families 7–11 with the mutation Ex7_8del showed the same haplotype between LIPH-MS1 and LIPH-MS4 (Figure S4). In 2.5 addition, Families 5 and 6 with the mutation 659-660delTA completely shared the same haplotype throughout all six LOD 1 markers analyzed, whereas only Family 4 with the same mutation showed a different haplotype from the other two

0.5 families (Figure S4).

LIPH mRNA is widely expressed in the hair follicle 0 In order to precisely localize the expression of LIPH within the HF, we performed in situ hybridization studies in –0.5 human scalp skin sections. In the bulb portion of the HF, 150 160 170 180 190 200 the LIPH mRNA is strongly expressed in the precortex, hair Mb shaft cuticle, and Huxley’s layer of the inner root sheath LIPH (Figure 3a and b). In upper portion of the HF, the signals Figure 2. Results of autozygosity mapping. The maximum LOD score within the outer root sheath becomes prominent (Figure 3c). (Z ¼ 2.00) was obtained for a region on chromosome 3q27.1–27.3. Furthermore, LIPH is also expressed in the sebaceous gland

624 Journal of Investigative Dermatology (2009), Volume 129 Y Shimomura et al. LIPH Mutations Underlie ARWH

Table 1. Summary of mutations in the LIPH gene Family Origin Main phenotype Exon Mutation Consequence Reference

Family 1 Pakistan WH 5 682delT FS/PTC This study Family 2 Pakistan WH 2 322T4C W108R This study Family 3 Pakistan WH/HT 4 624delT FS/PTC This study Family 4 Pakistan WH/HT 5 659–660delTA FS/PTC This study Family 5 WH/HT Family 6 HT Family 7 Pakistan WH 7, 8 Ex7_8del FS/PTC This study Family 8 WH/HT Family 9 WH Family 10 HT Family 11 HT Mari and Chuvash HT 4 Ex4del Deletion of 34 amino acids Kazantseva et al. (2006) populations in Russia Pakistan HT 2 346–350delATATA FS/PTC Ali et al. (2007) WH, woolly hair; HT, hypotrichosis; FS, frameshift; PTC, premature termination codon.

a bcHu

ORS

Hu Cu PC PC SG

d e f

Figure 3. LIPH mRNA is strongly expressed in the human HFs. Longitudinal sections at the (a, b, e) lower portion and (c) upper portion of the HFs. (d, f ) Interfollicular epidermis. (a–d) Antisense probe. (e, f) Sense probe. PC, precortex; Hu, Huxley’s layer; Cu; hair shaft cuticle; ORS, outer root sheath; SG, sebaceous gland. Scale bars: 100 mm(a, c, e), 20 mm(b, d and f ).

(Figure 3c) and the suprabasal layers of human epidermis tion suggested the existence of another gene associated with (Figure 3d). The sense probe did not show a positive signal ARWH. To find it, we performed linkage analysis in one of (Figure 3e and f). these families with the Affymetrix 10K array in order to identify the previously unknown causative gene for ARWH. DISCUSSION Unexpectedly, the family showed clear linkage to chromo- In this study, we analyzed 11 consanguineous Pakistani some 3q27, in the vicinity of the LIPH gene. families with ARWH, as well as other variable associated The human LIPH gene was previously cloned and features, including sparse and hypopigmented hair shafts. To characterized by two groups (Sonoda et al., 2002; Jin et al., our surprise, we did not find mutations in the P2RY5 gene of 2002). It encodes LIPH, which is a member of the these 11 families, despite the nearly identical phenotypes phosphatidic acid-selective phospholipase A1 protein family between these families and those we previously reported with (Sonoda et al., 2002). Similar to other lipase members, LIPH ARWH/hypotrichosis (Shimomura et al., 2008). This observa- protein has the N-terminal catalytic domain which contains

www.jidonline.org 625 Y Shimomura et al. LIPH Mutations Underlie ARWH

659–660delTA W108R 624delT 683delT Ex7_8del

S154 D178 β H248 NH2 S LD COOH

Ex4del (Kazantseva et al., 2006)

346–350delATATA (Ali et al., 2007)

108W C Human Chimpanzee Cow LIPH Dog Lid domain Mouse Frog β9 loop Zebrafish Catalytic Human LIPG W108 Human LIPC domain Human LPL Catalytic Human PS-PLA1 triad N

Figure 4. Schematic representation of LIPH protein and the LIPH mutations, and multiple amino-acid sequence alignment of LIPH of different species, as well as other human lipase members. (a) Mutations identified in this study are shown in red color above the schematic, and those reported previously (15, 16) are shown below. Three catalytic residues are indicated by asterisks. S, signal peptide; b, b9 loop; LD, lid domain. (b) Residues that are completely conserved between all members are colored in red, and those that are conserved among more than five members are colored in yellow. (c) A homology model of lipase H protein. The tryptophan residue at amino acid 108 (W108) within the catalytic domain is indicated in red color. Lid domain, b9 loop, and catalytic triad are also shown.

three catalytic residues at positions 154, 178, and 248 (Figure human lipase members (Figure 4b). We have generated a 4a) (Sonoda et al., 2002; Jin et al., 2002). In addition, it homology model of LIPH protein based on the crystal possesses a b9 loop and a lid domain, both of which are structure of the pancreatic lipase family members showing considered to be a crucial structure for substrate recognition high with LIPH (Figure 4c), which (Figure 4a) (Sonoda et al., 2002; Jin et al., 2002). Previously, suggests that the W108 side chain is buried and forms a only two homozygous mutations in the LIPH gene have been critical part of the hydrophobic core of the catalytic domain. identified in families with autosomal recessive forms of Substitution of the bulky hydrophobic side chain of trypto- hypotrichosis, specifically, Ex4del (Kazantseva et al., 2006) phan with the positively charged side chain of arginine would and 346–350delATATA (Ali et al., 2007). The mutation likely result in protein misfolding which would lead to Ex4del was identified as a founder in many families in the production of an inactive enzyme. Mari and Chuvash populations in Russia (Table 1; Figure 4a) As three and five families share the 659–660delTA and the (Kazantseva et al., 2006). The mutation 346–350delATATA Ex7_8del mutations, respectively, we analyzed the genotype was identified in a single consanguineous family of Pakistani of these families using microsatellite markers for the LIPH origin (Table 1; Figure 4a) (Ali et al., 2007). Although affected locus on chromosome 3q27 to determine if each mutation individuals with these LIPH mutations clearly showed sparse was carried on the same chromosome. In Families 7–11 with hair, neither report mentioned the WH phenotype (Kazantseva the mutation Ex7_8del, we found the same disease-related et al., 2006; Ali et al., 2007). Nevertheless, we focused on the haplotype in the region between LIPH-MS1 and LIPH-MS4 LIPH as a candidate gene for ARWH, as we postulated that that contains the LIPH gene (Figure S4), which indicates a hypotrichosis caused by LIPH mutations could be a founder chromosome for the mutation Ex7_8del. Similarly, phenotypic variant of WH, as we observed for P2RY5 affected individuals in Families 5 and 6 with the mutation (Shimomura et al., 2008). 659–660delTA are homozygous for the same haplotype Mutation analysis resulted in the identification of five throughout all six markers analyzed (Figure S4). Only affected homozygous mutations in the LIPH gene in all 11 families individuals in Family 4 with the same mutation 659–660del- analyzed (Figure S3). Four of these mutations, 624delT, TA showed a different haplotype from the other two families 659–660delTA, and 683delT, and Ex7_8del, result in a (Figure S4). This result may suggest the existence of another frameshift and a premature termination codon (PTC) (Table 1; founder for the 659–660delTA mutation, or that recombina- Figure 4a). Most likely, aberrant transcripts with a PTC would tion events have occurred over many generations, making the be largely degraded due to nonsense-mediated mRNA decay common region smaller than identified by the markers (Maquat, 1996; Frischmeyer and Dietz, 1999), leading to loss chosen. of expression of LIPH protein. The mutation W108R is an Recently, we reported that Pakistani families with P2RY5 amino-acid change within the N-terminal catalytic domain of mutations show not only WH, but also less dense or sparse LIPH. The tryptophan residue at amino acid 108 is highly hair, and such variations in phenotype are observed between conserved among LIPH of different species, as well as other families, as well as within a single family (Shimomura et al.,

626 Journal of Investigative Dermatology (2009), Volume 129 Y Shimomura et al. LIPH Mutations Underlie ARWH

2008). Consistent with these findings, mutations in the P2RY5 pathway in HF development and hair growth, which upon gene were reported in Saudi Arabian families with autosomal modulation may provide new targets for the treatment of recessive hypotrichosis simplex (OMIM 146520), which is excessive hair growth or hair loss. characterized early-onset paucity of scalp and body hair In this study, we have shown that mutations in the LIPH (Pasternack et al., 2008). Similarly, 11 families with LIPH gene underlie ARWH/hypotrichosis, in addition to the P2RY5 mutations identified in this study show a wide variation in gene. We have defined a role for a second gene involved in phenotype from WH to sparse hair between families and the pathogenesis of woolly and sparse hair, and provide even within a single family. One strikingly illustrative insights into determination of hair texture in humans. example is provided by the five families with the Ex7_8del mutation. Although these families share the identical muta- MATERIALS AND METHODS tion in the LIPH gene, Families 7 and 9 show only WH, Linkage analysis whereas Families 10 and 11 exhibit obvious sparse hair After obtaining informed consent, we collected peripheral blood (Figure S2). Furthermore, Family 8 shows variable severity samples from members of Pakistani families and 100 population- between affected individuals (Figure 1c and d). These matched unrelated healthy control individuals in EDTA-containing differences may be due to the influence of modifier genes, tubes (under institutional approval and in adherence to the genetic background, and/or environmental factors between Declaration of Helsinki Principles). Genomic DNA was isolated families, as well as among individuals within a family. from these samples according to standard techniques. A whole Collectively, these findings suggest that the phenotypes genome scan on 10 members of Family 1 was performed using the resulted from P2RY5 and LIPH mutations significantly over- Affymetrix 10K SNP array. Genespring GT (Agilent Software) was lap, and both mutations can show variable phenotypes from used for quality control measures and to perform a number of WH to sparse hair. We and others have previously reported analyses. Haplotypes were inferred from the data by Genespring GT. the same phenomenon of overlap between hypotrichosis and By using haplotypes, the effect of linkage disequilibrium on monilethrix (Kljuic et al., 2003; Shimomura et al., 2006; multipoint linkage analysis is minimized, reducing type I error. Schaffer et al., 2006; Zlotogorski et al., 2006). Such Initial analysis included whole-genome autozygosity mapping to observations emphasize the wide variability in the expression identify regions identical by descent that are shared among affected of hair phenotypes between different individuals (Schweizer, individuals. Details about the methodology employed by this test 2006; Sprecher, 2008). The key observation that unifies these can be found at https://www.chem.agilent.com/cag/bsp/products/ variable phenotypes is that all affected individuals with LIPH gsgt/Downloads/pdf/autozygosity.pdf. mutations in our Pakistani families have WH at birth and/or Parametric linkage analysis was performed using haplotypes and during early childhood, and some (but not all) progress to assuming a recessive mode of inheritance with 100% penetrance show different degrees of hypotrichosis. Therefore, we and a disease allele frequency of 0.001. conclude that the LIPH is a second causative gene for ARWH/hypotrichosis. Mutation analysis of the LIPH gene It has previously been shown that LIPH transcripts are Using the genomic DNA from members of Families 1–11, all exons of expressed in dissected human HF mRNA by reverse the LIPH gene with adjacent sequences of exon–intron borders were transcriptase–PCR (Kazantseva et al., 2006), however, amplified by PCR using gene-specific primers (Table S1). The the precise compartment within the HF was not defined. amplification conditions were 941C for 2 minutes, followed by 35 We performed in situ hybridization studies and showed that cycles of 941C for 30 seconds, 561C for 30 seconds, and 721Cfor the human LIPH mRNA is strongly expressed in the hair shaft, 50 seconds, with a final extension at 721C for 7 minutes. The amplified Huxley’s layer of the inner root sheath, and the outer root PCR products were directly sequenced in an ABI Prism 310 Automated sheath of the HF (Figure 3a–c). In addition, it is also expressed Sequencer, using the ABI Prism Big Dye Terminator Cycle Sequencing in sebaceous gland and the suprabasal layers of the epidermis Ready Reaction Kit (PE Applied Biosystems, Foster City, CA). In order (Figure 3c and d). We recently reported that P2RY5 are to detect the Ex7_8del mutation, we performed PCR using the primer expressed in the Henle’s and Huxley’s layers of the inner root pairs that were designed at intron 6 and intron 8 of the LIPH gene, sheath, as well as suprabasal layers of the epidermis respectively (Table S2). The amplification conditions were 941Cfor (Shimomura et al., 2008). Collectively, we have shown that 2 minutes, followed by 35 cycles of 941C for 30 seconds, 601Cfor the expression of human LIPH and P2RY5 overlap signifi- 30 seconds, and 721C for 30 seconds, with a final extension at 721Cfor cantly in Huxley’s layer of the HF inner root sheath and the 7 minutes. The amplified PCR products were analyzed on 1.5% epidermis. Importantly, it has been shown that LIPH is a key agarose gels, followed by direct sequencing. Screening assays for each enzyme in the synthesis of lysophosphatidic acid (LPA) from LIPH mutation were performed using genomic DNA from 100 phosphatidic acid (Sonoda et al., 2002). LPA is an extra- unrelated healthy control individuals (200 chromosomes) of Pakistani cellular mediator of many biological functions and has been origin following the protocols shown in Table S2. reported to promote hair growth in vivo (Takahashi et al., 2003). It has been previously reported that the nearest relative Genotyping of P2RY5, known as P2RY9/GPR23, is a receptor for LPA In order to analyze whether the mutations 659–660delTA and (Noguchi et al., 2003), and P2RY5 has recently been shown Ex7_8del in LIPH are founder mutations in Pakistan, genomic DNA to be a LPA receptor as well (Pasternack et al., 2008). These from affected individuals of Families 4–11 were amplified by PCR data suggest a crucial role of LIPH/LPA/P2RY5 signaling using primers for two microsatellite markers, D3S3592 and

www.jidonline.org 627 Y Shimomura et al. LIPH Mutations Underlie ARWH

D3S1602, close to LIPH gene, as well as four additional markers Jin W, Broedl UC, Monajemi H, Glick JM, Rader DJ (2002) Lipase H, a new (LIPH-MS1-4) around or within LIPH (Table S3). The amplification member of the triglyceride lipase family synthesized by the intestine. Genomics 80:268–73 conditions for each PCR were 941C for 2 minutes, followed by 35 Kazantseva A, Goltsov A, Zinchenko R, Grigorenko AP, Abrukova AV, cycles of 941C for 30 seconds, 551C for 30 seconds, and 721C for Moliaka YK et al. (2006) Human hair growth deficiency is linked to a 30 seconds, with a final extension at 721C for 7 minutes. PCR genetic defect in the phospholipase gene LIPH. Science 314:982–5 products were run on 8% polyacrylamide gels and genotypes were Kljuic A, Bazzi H, Sundberg JP, Martinez-Mir A, O’Shaughnessy R, Mahoney assigned by visual inspection. MG et al. (2003) Desmoglein 4 in hair follicle differentiation and epidermal adhesion: evidence from inherited hypotrichosis and acquired In situ hybridization pemphigus vulgaris. Cell 113:249–60 A part of the human LIPH cDNA (GenBank Accession number, Maquat LE (1996) Defects in RNA splicing and the consequence of shortened translational reading frames. Am J Hum Genet 59:279–86 NM_139248: nt. 1077–2360) was cloned into pCRII-TOPO vector McKoy G, Protonotarios N, Crosby A, Tsatsopoulou A, Anastasakis A, Coonar (Invitrogen, Carlsbad, CA). The antisense and sense DIG-labeled A et al. (2000) Identification of a deletion in plakoglobin in cRNA probes were synthesized from the linearized vectors with T7 arrhythmogenic right ventricular cardiomyopathy with palmoplantar and SP6 RNA polymerases (Roche Applied Science, Indianapolis, keratoderma and woolly hair (Naxos disease). Lancet 355:2119–24 IN), respectively. Tissues of human scalp skin were fixed with 4% Noguchi K, Ishii S, Shimizu T (2003) Identification of p2y9/GPR23 as a novel paraformaldehyde-PBS at 41C overnight, and processed to paraffin G protein-coupled receptor for lysophosphatidic acid, structurally distant from the Edg family. J Biol Chem 278:25600–6 sections. In situ hybridization was performed following the methods Norgett EE, Hatsell SJ, Carvajal-Huerta L, Cabezas JC, Common J, Purkis PE described previously with minor modifications (Aoki et al., 2001). et al. (2000) Recessive mutation in desmoplakin disrupts desmoplakin- intermediate filament interactions and causes dilated cardiomyopathy, CONFLICT OF INTEREST woolly hair and keratoderma. Hum Mol Genet 9:2761–6 All authors have no conflict of interest or financial interest in this work. Pasternack SM, von Ku¨gelgen I, Aboud KA, Lee YA, Ru¨schendorf F, Voss K et al. (2008) G protein-coupled receptor P2Y5 and its ligand LPA are ACKNOWLEDGMENTS involved in maintenance of human hair growth. Nat Genet 40:329–34 We gratefully acknowledge the families for having participated in this study. Salamon T (1963) U¨ ber eine familie mit recessiver Kraushaarigkeit, We thank Ha Mut Lam for excellent technical assistance. We appreciate Dr. hypotrichose und anderen anomalien. Hautarzt 14:540–4 Robert Bernstein for his insights and collaboration. This work was supported Schaffer JV, Bazzi H, Vitebsky A, Witkiewicz A, Kovich OI, Kamino H et al. by US Public Health Service National Institute of Health Grant R01AR44924 (2006) Mutations in the desmoglein 4 gene underlie localized autosomal from the National Institute of Arthritis and Musculoskeletal and Skin Diseases recessive hypotrichosis with monilethrix hairs and congenital scalp (to A.M.C.). erosions. J Invest Dermatol 126:1286–91 Schweizer J (2006) More than one gene involved in monilethrix: intracellular SUPPLEMENTARY MATERIAL but also extracellular players. J Invest Dermatol 126:1216–9 Table S1. Primers to amplify the LIPH gene. Shimomura Y, Sakamoto F, Kariya N, Matsunaga K, Ito M (2006) Mutations in Table S2. Primers and restriction enzymes used for screening assays. the desmoglein 4 gene are associated with monilethrix-like congenital hypotrichosis. J Invest Dermatol 126:1281–5 Table S3. Primers for additional microsatellite markers. Shimomura Y, Wajid M, Ishii Y, Shapiro L, Petukhova L, Gordon D et al. Figure S1. Pedigrees and clinical features of Families 1–4. (2008) Disruption of P2RY5, an orphan G protein-coupled receptor, Figure S2. Pedigrees and clinical features of Families 5–11. underlies autosomal recessive woolly hair. Nat Genet 40:335–9 Figure S3. Identification of mutations in the LIPH gene. Sonoda H, Aoki J, Hiramatsu T, Ishida M, Bandoh K, Nagai Y et al. (2002) A Figure S4. Haplotype of affected individuals of Families 4–11 in the LIPH novel phosphatidic acid-selective phospholipase A1 that produces locus. lysophosphatidic acid. J Biol Chem 277:34254–63 Sprecher E (2008) Disentangling the roots of inherited hair disorders. Nat REFERENCES Genet 40:265–6 Stratigos AJ, Baden HP (2001) Unraveling the molecular mechanisms of hair Ali G, Chishti MS, Raza SI, John P, Ahmad W (2007) A mutation in the lipase and nail genodermatoses. Arch Dermatol 137:1465–71 H (LIPH) gene underlie autosomal recessive hypotrichosis. Hum Genet 121:319–25 Takahashi T, Kamimura A, Hamazono-Matsuoka T, Honda S (2003) Phosphatidic acid has a potential to promote hair growth in vitro and Aoki N, Sawada S, Rogers MA, Schweizer J, Shimomura Y, Tsujimoto T et al. in vivo, and activates mitogen-activated protein kinase/extracellular (2001) A novel type II cytokeratin, mK6irs, is expressed in the Huxley signal-regulated kinase kinase in hair epithelial cells. J Invest Dermatol and Henle layers of the mouse inner root sheath. J Invest Dermatol 121:448–56 116:359–65 Weeda G, Eveno E, Donker I, Vermeulen W, Chevallier-Lagente O, Taı¨eb A Chavanas S, Bodemer C, Rochat A, Hamel-Teillac D, Ali M, Irvine AD et al. et al. (1997) A mutation in the XPB/ERCC3 DNA repair transcription (2000) Mutations in SPINK5, encoding a serine protease inhibitor, cause gene, associated with trichothiodystrophy. Am J Hum Genet 60:320–9 Netherton syndrome. Nat Genet 25:141–2 Winter H, Rogers MA, Langbein L, Stevens HP, Leigh IM, Labre`ze C et al. Chien AJ, Valentine MC, Sybert VP (2006) Hereditary woolly hair and (1997) Mutations in the hair cortex keratin hHb6 cause the inherited hair keratosis pilaris. J Am Acad Dermatol 54:S35–9 disease monilethrix. Nat Genet 16:372–4 Frischmeyer PA, Dietz HC (1999) Nonsense-mediated mRNA decay in health Zlotogorski A, Marek D, Horev L, Abu A, Ben-Amitai D, Gerad L et al. (2006) and disease. Hum Mol Genet 8:1893–900 An autosomal recessive form of monilethrix is caused by mutations in Hutchinson PE, Cairns RJ, Wells RS (1974) Woolly hair. Clinical and general DSG4: clinical overlap with localized autosomal recessive hypotricho- aspects. Trans St Johns Hosp Dermatol Soc 60:160–77 sis. J Invest Dermatol 126:1292–6

628 Journal of Investigative Dermatology (2009), Volume 129