Mapping and DNA sequence analysis of Genes underlying Isolated Autosomal Human Hereditary Alopecia in Pakistani families

by Khushbakht Khan (Author) Sehrish Khan (Author)

Bachelor Thesis 2014 84 Pages

Biology - Micro- and Molecular Biology



List of Figures

List of Tables

List of Ellipsis


1. Chapter 1 : Introduction Introduction
1.1. Hair composition and structure
1.2. Hair follicle morphogenesis
1.3. Hair follicle cycling:
1.4. Types of Hair Disorder/Alopecia
1.4.1. Complex Hair Disorders Androgenic Alopecia Alopecia Areata
1.4.2. Syndromic Alopecia Alopecia with mental retardation syndrome Hypotrichosis with juvenile macular dystrophy Netherton Syndrome Human Nude Phenotype
1.4.3. Inherited Isolated Alopecias Inherited Isolated Autosomal Dominant Alopecias Inherited Isolated Autosomal Recessive Alopecias
1.5. Genetics of Alopecia

2. Chapter 2: Materials and Methods
2.1. Families studied
2.2. Pedigree Analysis
2.3. Extraction of Genomic DNA
2.3.1. Phenol-Chloroform Method
2.3.2. Thermo scientific commercially available kit
2.4. Polymerase Chain Reaction
2.5. Linkage Analysis
2.6. Gel Electrophoresis
2.6.1. Agarose Gel Electrophoresis
2.6.2. Polyacrylamide Gel Electrophoresis (PAGE)
2.7. Gene Sequencing
2.7.1. Genomic DNA Amplification for Sequencing First sequencing PCR First Purification Second sequencing PCR Second Purification
2.8. Mutation analysis

3. Chapter 3: Results
3.1. Family A
3.2. Family B
3.3. Genetic mapping of candidate genes for autosomal recessive alopecia
3.4. CDH3 gene sequencing

4. Chapter 4: Discussion

5. Chapter 5: References

List of Figures

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List of Tables

Table 1.1 Classification of Alopecia

Table 2.1 Composition of different solutions used

Table 2.2 List of microsatellite markers used to test linkage to genes involved in alopecia.

Table 2.3 List of primer sequences used to amplify CDH3 gene exons

List of Ellipsis

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Alopecia is a broad term including many forms of hereditary hair loss resulting from genetic defects affecting hair growth cycle or hair structure that vary in age of onset, severity and associated ectodermal abnormalities. The inheritance pattern of alopecia can be autosomal dominant, autosomal recessive or X-linked. Various mutations in several genes on different chromosomes are being identified which are involved in pathogenesis of inherited autosomal recessive alopecia.

In present research, two families (A;B) with isolated hereditary alopecia, residing in different zones of Pakistan were ascertained. The mode of inheritance inferred as autosomal recessive. One family was subjected to mutation screening while on other, polymorphic microsatellite markers was used for the purpose of homozygosity mapping to explicate the gene defect. Phenotypic analysis of family A shows the characteristic clinical features of hypotrichosis with sparse hair on head and rest of body and with no associated abnormality. Gene linked to this family in previous research was CDH3. So, splice-junction site and sixteen exons of this gene were sequenced but were negative for functional sequence variant. This clearly shows mutation must be present in regulatory region of this gene.

In family B, affected individual’s shows clinical features of atrichia with papular lesions (APL) which is rare autosomal recessive disorder, characterized by occurrence of complete hair loss with the development of keratin-filled cysts. Known candidate genes (DSG4, HR, LIPH and LPAR6) were tested for homozygosity mapping via polymorphic microsatellite markers. Genotyping data showed no linkage to any of the candidate loci and therefore, their involvement in causing atrichia with papular lesions in this family is not supported.


Alopecia is a general term referring to collection of human hereditary hair loss phenotypes. Most of the phenotypes of alopecia manifest due to disruption in morphology or physiology of hair follicles. There are many types of alopecia which can be classified on the basis of presence or absence of different signs and symptoms, severity, age of onset, associated ectodermal abnormalities including teeth, nails, sweat glands and brain and pattern of inheritance (Wali et al., 2007a). Alopecia appear in variety of patterns like alopecia areata is characterized by localized patches of hair loss, reticular pattern has innumerable small patches that coalesce, alopecia totalis is complete hair loss of scalp and entire hair are lost from all over the body in alopecia universalis (Fenton, 2004). There are at least 50 types of congenital hair loss disorders that are inherited as isolated or syndromic form (Shimomura, 2012).

Its prevalence is highly dependent upon the age and sex of the affected individuals. In Iran, around 39.6% women belonging to different age groups are reported affected with this disease (Fatemi et al., 2010). Survey in USA showed pattern hair loss in 53% of men (age >44) population (Rhodes et al., 1998). In Korean population, it is reported in 14.1% of men (Paik et al., 2001). In Singaporean males, its prevalence is reported 63% (Tang et al., 2000). Prevalence of alopecia in Pakistani population has not been estimated yet. However, in Asians, it is reported to be 3.78% of dermatologic patients (Shamsadin et al., 2006).

1.1. Hair composition and structure

Hairs are present almost all over the body surface except on the palms, soles and sides of finger and toes. They are filamentous structures, composed of keratin which is a structural protein and constitutes 65-95% of hair protein (Powell and Rogers, 1997).

Hair consists of two major parts: shaft and follicle. Hair shaft (HS) grows outside the scalp. It consists of three distinct layers; cuticle, cortex and medulla. All three layers contribute in the appearance of the shaft by influencing its shape and structure. Inner most layer is called medulla which is only found in large think hair. Then, the middle layer is cortex and is a major site for kératinisation (Langbein et al., 2001). Thus, it is vital for shaft rigidity and strength. This layer also provides hair its texture and color. Outer layer cuticle forms the hair surface and protects cortex (Hardy, 1992).

While, hair follicle is located in dermal layer of the skin and is associated with sebaceous glands. It can be divided in to four areas; infundibulum, isthmus, lower follicle and bulb. It is a complex epithelial structure, surrounded by concentric sheaths, performing distinct functions (Langbein and Schweizer, 2005). Outer root sheath (ORS) is the outer most non-keratinizing layer of the follicle and has no role in hair growth; instead, it plays its part in protecting growing hair. Then comes the companion layer and inner root sheath (IRS) which spread from outside to inside. It consists of three compartments; layers of Henley, layers of Huxley and IRS cuticle. Dermal papilla (DP) is present at the base of HF and contains numerous blood vessels and sensory nerves. Hair bulb resides with in DP and growing hair originates from here. Hair bulge is also found within HF and it is a site for epithelial and melanocytic stem cell (reviewed in Lai-Cheong and McGrath, 2013).

1.2 Hair follicle morphogenesis

Hair morphogenesis begins during the 10th week of gestation (Stenn and Paus, 2001) from the single uniform layer of multipotent ectoderm. These undifferentiated cells are destined to differentiate into two main cell lineages, epidermal and hair. In order to create protective layer, ectoderm cells of epithelial lineage detaches from basement layer and moves toward skin surface where it creates keratinized, squamous cell layer (Fuchs and Raghavan, 2002; Dai and Segre, 2004) (Figure 1.1).

While, on receiving mesoderm derived Wnt signals, these multipotent cells gave rise to primordial hair follicle bud (Lee and Tumbar, 2012). This embryonic hair morphogenesis initiates with the formation of placodes which are composed of mesodermal cells and are formed by the localized thickening of surface ectoderm which is preceded by the bud like invagination into dermis. Wnt/ß catenin signaling (reviewed in S.-Y. Tsai et al., 2014) and epithelial ectodysplasin EDA/EDAR signaling are mainly involved in placode formation (Sennett and Rendl, 2012). Then, signaling from placode leads to formation of dermal condensate just below the placode which results in hair germ formation. This HG further pass signals for proliferation and invaginates into dermis to form hair peg and cause condensate mesenchyme to become dermal papilla (DP) (Millar, 2002; Lee and Tumbar, 2012).

As further growth proceeds, DP signals epithelially derived matrix cells, surrounding DP to form hair bulb (Paus et al., 1999). Further exchange of epithelial-mesenchymal signals induces matrix cells terminal differentiation in to inner root sheath (IRS) and hair shaft (Paus et al., 1999; Sennett and Rendl, 2012). Those cells that lose contact with hair bulb differentiate into outer root sheath (ORT) (Hardy, 1992).

First hair in human embryo is called lanugo hair. Lanugos are thin, soft, non-medullated and usually unpigmented and are shed before the birth and are replaced by finer, short colorless hair called, Vellus hair. Vellus are difficult to see and are replaced by terminal hair which remains present in adulthood as well.

1.3 Hair follicle cycling

After the HF morphogenesis, the HFs undergoes dynamic cell kinetics, known as hair cycles (Shimomura and Christiano, 2010). This follicle regeneration occurs throughout the postnatal life and is maintained by multipotent HF stem cells which are present primarily in the bulge region of the outer root sheath (Cotsarelis et al., 1990). These multipotent HF stem cells are also found in the lower parts of whisker HFs (Claudinot et al., 2005). This cycling consists of following phases (Figure 1.1).

1. Anagen: It is a growing phase and is characterized by the beginning of cell proliferation of multipotent HF stem cells (Oshima et al., 2001) and their migration from bulge region to matrix region. Which is further accompanied by matrix cells extensive proliferation and differentiate into hair shaft and inner root sheath (Lee and Tumber, 2012). Melanocytes are located proximal to forming hair shaft and melanin synthesis occurs in this phase in pigmented animals (Slominski and Paus, 1993). This lasts for 2-6 years in humans (Oshima et al., 2001) and hair grows approximately 10cm in this duration.

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Figure 1.1 Schematic diagram of different stages of follicular morphogenesis and the hair cycle. HF is specified from the epidermis only during embryogenesis and enters the postnatal hair cycle, where follicles go through stages of growth (anagen), regression (catagen) and rest (telogen). Only the lower portion of the hair follicle below the bulge is regenerated with each hair cycle (Paus and Cotsarelis, 2004).

2. Catagen: After the growth phase, the regression phase begins. The proliferation and differentiation of HF reduces dramatically and HF production ceases (Stenn and Paus, 2001) due to the apoptosis/programmed death of the matrix cells, differentiated layers and lower follicle’s epithelial layer. Dermal papilla does not undergo apoptosis and remain intact (Lee and Tumber, 2012). The duration of this phase is 1-2 weeks (Oshima et al., 2001).

3. Telogen: After apoptosis, follicle enters in to resting phase which in humans, lasts for about 2-3 months (Oshima et al., 2001). This phase is characterized by the minimum signal exchange between the dermal papilla and karatinocytes (Stenn and Paus, 2001). After it, dermal papilla again signals stem cells for proliferation and new hair follicle regeneration cycle begins.

4. Exogen: Sometimes after entering into telogen, regeneration cycle does not begin again and hair sheds (Stenn et al, 1998).

Duration of each phase determines the length of hair while duration of each phase varies from individual to individual and also varies depending upon body site. For instance, in human scalp, mostly follicles are in anagen phase lasting for 2-6 years while catagen lasts only for few weeks and telogen for 3 months (Krause and Foitzik, 2006).

1.4 Types of Hair Disorder/Alopecia

Alopecia is classified on the bases of inheritance fashion and association with other ectodermal abnormalities as isolated, syndromic and complex alopecia. These types are summed up in table 1.1.

1.4.1 Complex Hair Disorders

Common genetic disorders that are demonstrable under polygenic phenomenon are called complex hair disorders. Affected individuals show marked variation in disease phenotypes. Androgenic Alopecia

Androgenic alopecia (AGA; MIM 109200) is an androgen dependent hereditary disorder of hair with defined pattern of progressive hair thinning. Androgenetic alopecia also referred to as male pattern baldness (MPB) and as female pattern baldness (FPB) in men and women respectively. It affects at least 50% of men by the age of 50 years and up to 70% of all males in later life (Tully et al., 2010). Alopecia Areata

Alopecia areata (AA; MIM 104000) is one of the most common autoimmune, non­scarring, inflammatory scalp and body hair disorder. It has sudden onset with unpredictable progression and recurrent throughout life. All individuals have approximately 2% lifetime risk irrespective of gender and ethnic group (Gilher and Kalish, 2006). Patchy hair loss on the scalp involving the entire scalp is known as alopecia totalis. If the entire body is involved then this is called alopecia universalis (Fenton, 2004). The patch in this alopecia usually has a characteristic border where normal hair demarcates the margins of the lesion. The number of hair follicles appears to remain the same in the initial stages of this disease, however, this number decrease and miniaturization of the anagen hair follicles is observed in the more advanced stages (McDonagh and Messenger, 1996).

1.4.2 Syndromic Alopecia

Syndromic alopecia is associated with other clinical conditions including mental, ocular, cardiac, immunodeficiency, erythrodermal, ectodermal and its appendages. Such types of alopecia follow both autosomal dominant and recessive mode of inheritance. Alopecia with mental retardation syndrome:

Alopecia with mental retardation syndrome (APMR; MIM 203650) is inherited in autosomal recessive fashion with total or partial hair loss (scalp, eyebrows, eyelashes, axillary, and pubis hair) and mild to severe mental retardation. Patients have normal nail, teeth, hearing and sweating processes (John et al., 2006a). Hypotrichosis with juvenile macular dystrophy

Hypotrichosis with juvenile macular dystrophy (HJMD; MIM 601553) a rare hereditary alopecia characterized by progressive macular degeneration which leads to blindness during the second decade of life (Yasakura et al., 1967). Netherton Syndrome

Netherton syndrome (NS; MIM 256500), first time reported by Netherton in 1958, is severe autosomal recessive hair disorder with high postnatal mortality. It is characterized by congenital ichthyosiform erythroderma, trichorrhexis invaginata (hair shaft defect) and severe atopic manifestation. Sparse and brittle hair upon microscopic express reveals nodes called trichorrhexis invaginata or bamboo hair form due to invagination of the distal part to proximal part of hair shaft (Netherton, 1958). Human Nude Phenotype

Human Nude Phenotype is characterized by congenital alopecia, intrinsic thymus defect and nail dystrophy (Adriani et al., 2004).

1.4.3 Inherited Isolated Alopecia

Inherited isolated alopecia is condition of hair loss, hypotrichosis or woolly hair without involvement of other ectodermal appendages abnormalities. Isolated alopecia can be inherited in autosomal dominant, autosomal recessive and some in X- linked fashion (Al- fouzan ; Nanda, 2001). Inherited Isolated Autosomal Dominant Alopecia


Monilethrix (MIM 158000), an autosomal dominant form of alopecia is characterized by beaded appearance. It is a congenital hair shaft defect and inherits in fully penetrant form with variable expressions. Affected individuals have normal hair at birth but normal hair are replaced by brittle and fragile hair within first few months of life that easily fracture at narrow internodes and produce bald patches. Mild conditions affect only scalp including occipital area and nape of the neck while sever forms involve secondary sexual hairs, eyebrows and eyelashes (Narmatha et al., 2002).

Marie Unna Hereditary Hypotrichosis

Marie Unna hereditary hypotrichosis (MUHH; MIM 146550), a rare autosomal dominant alopecia, was reported by a German dermatologist Marie Unna in 1925 for the first time (Unna, 1925). It is characterized by coarse, wiry and twisted hair production in childhood that shed near puberty from vertex and scalp margins resulting in patchy to complete hair loss. Eyebrows, eyelashes, axillary, pubic and body hair are spares to absent (Sreekumar, 2000).

Hypotrichosis simplex

Hypotrichosis simplex (MIM 605389), another rare autosomal dominant alopecia, is characterized by sparse and thin hair. Reduced hair growth affects scalp and body hair however eyebrows, eyelashes and beard hair remain normal. Hypotrichosis simplex is inherited in isolated fashion and overall psychomotor development of affected individuals is normal (Baumer et al., 2000).

Hypotrichosis simplex of scalp

Hypotrichosis simplex of scalp (HTSS; MIM 146520) is an autosomal dominant isolated alopecia. Individuals affected by this form of alopecia are born with normal hair but in middle of first decade experience progressive scalp hair loss. However no abnormalities of body hair, beard, eyebrows, eyelashes, axillary hair, teeth and nail are observed.

Dominant Hereditary Hypotrichosis

Dominant Hereditary Hypotrichosis (MIM 094300) is mostly manifested with woolly hair following autosomal dominant fashion. In this disease individuals are born with coarse, lusterless, dry and tightly curled hair with normal density (Shimomura et al., 2010).

Table 1.1 Classification of Alopecia.

illustration not visible in this excerpt Inherited Isolated Autosomal Recessive Alopecia

Atrichia with Papular Lesions

Atrichia with Papular Lesions (APL; MIM 209500) is an autosomal recessive form of alopecia. It is characterized by presence of papules and cornified, material filled follicular cysts on scalp skin with no other defect of ectodermal appendages like nails, sweat gland and teeth. Normal hair are present at the time of birth that shad off during first month of life. Affected individuals have no scalp, axillary and body hair and are completely devoid of eyebrows and eyelashes (Ahmad et al., 1993; Ahmad et al., 1998a).

Localized Autosomal Recessive Hypotrichosis

Localized Autosomal Recessive Hypotrichosis is a rare type of alopecia caused by abnormality in hair follicles. It is histologically identified by atrophic and thin shafts that are coiled up within the skin due to their inability to penetrate the epidermis. Hairs are present on the scalp at birth but regrow sparsely after ritual shaving. Three types of autosomal recessive hypotrichosis have been reported to date.

- LAH1: In localized autosomal recessive hypotrichosis type 1 (LAH1; MIM 607903) hair loss is restricted to the scalp, chest, arms and legs with less dense facial hair, including eyelashes, eyebrows and beard, and sparse axillary and pubic hair. Affected individuals have normal growth and development like hearing, nail, teeth and sweating mechanisms. LAH1 is mapped on chromosome 18q12.1 with causative gene located in Desmoglein gene cluster (Kljuic et al., 2003; Rafique et al., 2003).

- LAH2: Localized autosomal recessive hypotrichosis type 2 (LAH2; MIM 609167) is characterized by sparse hair on the scalp at the time of birth that do not regrow after ritual shaving. Affected individuals have sparse or woolly hair with no eyebrows, eyelashes, axillary and body hair but normal nails and teeth. Affected male individuals have normal beard. LAH2 is mapped on chromosome 3q26.33-q27.3 with LIPH being main candidate gene (Aslam et al., 2004).

- LAH3: Localized autosomal recessive hypotrichosis type 3 (LAH3, MIM 611452) is caused by mutation LPAR6 gene. The patients exhibit typical features of the hereditary hypotrichosis and/or woolly hairs (Wali et al., 2007b).

1.5 Genetics of Alopecia

In depth molecular genetic studies of alopecia has revealed that many loci contribute in normal hair cycling and mutation in any of the loci result in manifestation of diseased phenotypes. Five autosomal dominant forms of isolated alopecia have been mapped on different chromosomes with genes known in majority of cases. Similarly, eight autosomal recessive forms of non-syndromic alopecia have been reported to arise as a result of mutations in following genes.

Hair less (HR) gene located on chromosome 8q22-21, spanning more than 14 kb, encodes for single zinc finger transcription factor which acts as a regulator during the transition of catagen phase into anagen phase in HF cycle (Panteleyev et al., 1999). Abnormality in it results in loss of HR protein function which causes the complete hair loss (Ahmad et al., 1998) via the loss of contact between the follicular epithelium and the dermal papilla (Panteleyev et al., 1999). Mutations in this gene is reported associated with Marie Unna hypotrichosis (Unna, 1925), alopecia universal and Atrichia with Papular Lesions (APL) (Ahmed et al., 1998).

It consists of 19 exons and mutations in this gene usually lie in its exons or in exon-intron boundary sequences. Autosomal dominant alopecia MUH is reported associated with this gene but initial studies failed to identify the mutation associated with this condition (van Steensel et al., 1999). Later, studies identified mutations in upstream ORF region of its promoter (Wen et al., 2009). c.3G>A U2HR mutation in German MUHH family is also identified (Redler et al., 2011). Further, 14 different mutations including single base substitution and two non sense mutations, have been reported in 19 MUHH families of various ethnic backgrounds (Du" zenli et al; Wen et al., 2009) and in one Chinese family (Cai et al., 2009). In case of APL, three non sense mutation: C-to-T transition (CAG- TAG) in the glutamine residue at position 323, changing to a stop codon (Q323X) in exon 3, C-to-T transition (CAG-TAG) causing Q502X in exon 6 and in exon 14 C-to-T transition (CAG-TAG) causing R940X, are reported in one study in Pakistani families (Kim et al., 2007). The effect of these mutations might be on mRNA stability (Maquat, 1996).A missense mutation in third exon of the HR gene (c.974G/A, p.Gly325Asp) is identified in Hungarian family in which affected individual was suffering from limb deformity along with MUH (Farkas et al., 2012). Two non sense mutations p.Cys690X and p.Arg819X, causing instability of truncated protein and a homozygous missense mutation (c.3470C>G) is also reported in Pakistani families (Azeem et al., 2011). Apart from those 43 mutations including 12 deletions, four insertions, 14 nonsense, eight missense and five splice-sites in the HRgene have been reported (Azeem et al., 2011).

Intracellular junctions provide infrastructure for signal transmission that is critical for orchestrating molecular events during proliferation and differentiation of HF. Cell to cell adhesion in vertebrate epithelial cells is mediated by desmosomes that are elaborate multi-protein complexes that connect cadherin partners to intermediate filaments network. Desmogleins and desmocollins are the glycoproteins of desmosomes (Fuchs et al., 2001). Desmoglein gene is mapped on chromosome 18q12 in the form of cluster of four genes (DSG1, DSG2, DSG3 and DSG4) (Whittock and Bower, 2003). Only desmoglein 4 is present in the inner layer of the hair follicles that suggests a critical role for desmoglein 4 in differentiation of ascending hair follicle layer. DSG4 gene is composed of 16 exons spanning approximately 37 kb of genomic DNA and is situated between DSG1 and DSG3.

Mutations in DSG4 result in localized autosomal recessive hypotrichosis type 1 (LAH1). Kljuic et al. (2003) identified an identical homozygous 5-kb deletion (EX5_8del) from 35 nucleotides upstream of exon 5 to 289 nucleotides downstream of exon 8 within the DSG4 gene in affected individuals from 2 consanguineous Pakistani families with localized autosomal recessive hypotrichosis. The mutation caused disturbance in dimerization domain of protein which is necessary for proper cell-cell adhesion. A single nucleotide deletion (87delG) within exon 3 resulted in frame shift and premature termination 162 bp downstream of the deletion leading to absence of functional DSG4 in patients (Wajid et al., 2007). Two heterozygous mutations have been identified in Japanese families causing monilethrix-like congenital hypotrichosis. One was a heterozygous T-C transition (574T-C) in exon 6 of the DSG4 gene and an S192P substitution at the protein level, resulting in abolished EcoRI restriction enzyme site. Other was a heterozygous 1bp (T) insertion missense mutation (2039insT) in exon 13. This frame shift lead to premature stop codon downstream of the mutation (Shimomura et al., 2006). Compound heterozygous splice site and a missense mutation in the DSG4 gene in affected Iraqi and Iranian Jewish individuals resulted in monilethrix-like congenital hypotrichosis. 216+1G>T in intron 3 and a C-to-G transversion at nucleotide position 800 in exon 7 disrupts a conserved calcium-binding site in the extracellular (EC)2-EC3 interface (Schaffer et al., 2006).

Lipase H (LIPH) gene, located on LAH2 locus on chromosome 3q26.33 is 45 kb gene (Sonoda et al., 2002) is another candidate loci for ARA. It codes for 451 amino acid protein which is membrane-associated phosphatidic acid-selective phospholipase A1 (mPA-PLAla) and catalyzes the production of LPA (Jin et al., 2002) by hydrolyzing PA in to LPA (Sonoda et al., 2002). The 55kDa protein product of this gene has N-terminal signal sequence followed by a catalytic domain containing catalytic triad Ser 154, Asp178, and His 248. Further, it contains three surface loops: the lid loop, the b5 loop, and the b9 loop that cover the active site and four potential N-linked glycosylation sites (Sonoda et al., 2002).

It consists of 10 exons and abnormalities in it are associated with ARWH/HT phenotype (Kazantseva et al., 2006) in families of different geographical backgrounds. Different mutations in its exons are reported up till now. For instance, a recurrent duplication mutation c.280_369dup is reported in exon 2 of Israeli family. This mutation adds 30 amino acids to the protein sequence but do not have any effect on reading frame (Horev at al., 2009). Similar mutation is also reported in three Arab families from Israel and one family from Turkey (Nahum et al., 2009). Horev and colleagues also reported deletion/insertion mutation [c.620_627 delACACTGATinsCTCCTTTCCTTGTG] in exon 4 in an Italian family which tends to replace three amino acids: Asp Thr Asp in position 207-209 with five amino acids: Ala Pro Phe Lue Val. This insertion again do not affects reading frame but the nature of substituted amino acids differ from original amino acids (Horev at al., 2009). 5bp deletion mutation (c.346-350delATATA) in exon 2 of the

Pakistani family is reported (Ali et al., 2007). This causes the frame shit and downstream premature termination codon, leading to instability of the protein. Then, two missense mutations c.2T > C in exon 1 and c.322T > C in exon 2 are reported in two consanguineous Pakistani families (Naz et al., 2009). First missense mutation might disrupt the initiation code (pM1T) (Naz et al., 2009) which causes the reduction in LIPH protein amount (Naqvi et al., 2009) while the second mutation causes the substitution of evolutionary conserved aromatic tryptophan by a polar arginine (Naz et al., 2009) which might cause abnormality in protein secondary structure. Another missense mutation c.736T>A (p.C246S) in its exon 6 in Japanese family is also reported (Shinkuma et al., 2010; Yoshizawa et al., 2013) which damages the functionality of PA-PLAla protein (Shinkuma et al., 2010). c.699C>G missense mutation in exon 5 of this gene is also reported in Japanese family (Yoshizawa et al., 2013). The affect of which is still unknown. A homozygous splice site mutation (IVS4-lG^C) is also reported in Pakistani family which alters the splice acceptor site of intron 4 and may results in skipping of exon 5 (Kalsoom et al., 2010). Then a recurrent mutation (c.659-660delTA) is identified in Pakistani family which altered the reading frame for ß9 loop (Kalsoom et al., 2010). In recent study, a non-sense mutation (p. Arg 110*) is detected which leads to premature termination codon in exon 2 whose result may be protein instability with the loss of catalytic triad (Mehmood et al., 2014).

Lysophosphatidic acid receptor 6 (LPAR6) gene or P2RY5 codes into a 344 amino acids long G-protein coupled receptor P2RY5 and is present on chromosome 13q14.11-q2l.32 (Azhar, et al., 2012). LPA produced by the action of LIPH protein (Harel ; Christiano, 2012) is a ligand for P2Y5 (Pasternack et al., 2008). LPA is actually a mixture of variety of fatty acids and is a key player in many cellular processes like cell proliferation, cell migration, apoptosis and smooth muscle contraction among others (Khan et al., 2011). Mutations in LPAR6 results in third class of hypotrichosis with autosomal recessive pattern of inheritance known as localized autosomal recessive hypotrichosis (LAH3) (Mahmoudi et al., 2012). Correct expression of LPAR6 and proper G-protein coupled receptor path way is responsible for maintenance of hair texture and hair growth cycle. Some mutations in the gene can either result in complete hair loss or woolly hair (Khan et al., 2011). Many mutations have been reported till date among which some are, a 12-kb deletion resulted in triple barrel structure due to illegitimate recombination causing strand slippage (Mahmoudi et al., 2012), A homozygous 1 base pair deletion (c.472delC) in Turkish patient and a four base pair duplication (c.64_67dup-TGCA) have been reported to cause localized autosomal recessive hypotrichosis and woolly hair (Pasternack et al., 2009). A study on 22 unrelated Pakistani families reported following novel mutations: a G to C transition at nucleotide position 8 (c.8G > C), caused substitution of serine with threonine (p.S3T), c.36insA (p.D13RfsX16) insertion of nucleotide A at position 36 caused frame-shift and premature termination codon 8 bp downstream , similarly insertion of nucleotide A at position 160 (c.160insA and p.N54TfsX58) resulted in frame-shift and premature termination codon 10 bp downstream the gene and a single homozygous base pair substation (c.436G > A and p.G146R) at position nucleotide 436 caused replacement of glycine with arginine (Azeem et al., 2008).

cadherin 3 (CDH3) gene is one more emerging loci for linkage analysis of this disease. This gene encodes a classical cadherin molecule, P-cadherin which is responsible for calcium-dependent cell-cell adhesion. P-cadherin is expressed in the retinal pigment epithelium and also in the hair matrix. CDH3 is 55 kb long gene comprising of 16 exons resides on chromosome 16q22.1, and comprised of 16 exons (Xu et al., 2002).

Sprecher e al. (2001) has reported hypotrichosis with juvenile macular dystrophy caused by mutated CDH3 in four consanguineous families of northern Israel. A homozygous deletion was found in all four families. Kjaer et al. (2005) studied 2 families with ectodermal dysplasia, ectrodactyly, and macular dystrophy and identified homozygous mutations in CDH3 in affected individuals. In family 1, a missense mutation (c.965ART) causes a change of amino acid 322 from asparagine to isoleucine. This replacement of amino acid affects Ca2+ binding affecting specificity of the cell-cell binding function. In family 2, a homozygous frame shift deletion (c.829delG) introduces a truncated fusion protein with a premature stop codon at amino acid residue 295. This mutation produces non-functional protein lacking both its intracellular and membrane spanning domains and its extracellular cadherin repeats.

Recently Basit et al. (2011) showed involvement of CDH3 in isolated autosomal recessive hypotrichosis in two Pakistani families. The sequence analysis revealed a single base pair homozygous insertion (c.1024_1025insG and p.342insGfsX345) in exon 9 in one family and a deletion of four nucleotides (c.1859_1862delCTCT and p.620delSfsX629) in exon 13 of the CDH3 gene in other family. Further fine mapping and sequencing work are required in order to identify the modifier gene help to understand the mechanism underlying the role of epistatic interactions in phenotypic variability.

In present study, two Pakistani families with inherited Alopecia have been ascertained. The main aim is to unveil the loci/genes in the studied families and novel sequence variants in the linked genes, as well as and molecular genetic analysis of phenotypes segregating in these families. Linkage analysis and homozygosity mapping was performed by genotyping different microsatellite markers to localize the disease genes in these families. DNA sequencing of the candidate genes has performed to identify the pathogenic mutation underlying the disease.

This study includes defining the inheritance pattern, clinical phenotypes and molecular genetic studies. The data presented here include identification of the disease causing gene and mutational analysis of CDH3gene. The main objectives of this research are:

- To determine the phenotypic and genetic variability in alopecia families from Pakistan.
- To identify the loci/genes responsible for the alopecia in Pakistani population.
- To perform Sequencing Analysis for finding the causative mutation in the specific genes.



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mapping genes isolated autosomal human hereditary alopecia pakistani




Title: Mapping and DNA sequence analysis of Genes underlying Isolated Autosomal Human Hereditary Alopecia in Pakistani families