Forensic Examination of Hair (1998) PDF

1 Physiology and Growth of Human Hair HARRY HARDING and GEORGE ROGERS 1.1 Introduction Hair is a characteristic of mammalian skin. On some animals it serves to keep them warm, or cool, to protect them from the weather and hazards of the environment, or to camouflage them as protection from at...

1 Physiology and Growth of Human Hair HARRY HARDING and GEORGE ROGERS 1.1 Introduction Hair is a characteristic of mammalian skin. On some animals it serves to keep them warm, or cool, to protect them from the weather and hazards of the environment, or to camouflage them as protection from attack by their predators. For the human animal these functions are not really necessary, and hair seems to be a dilemma. If we’ve got it, it’s in the wrong place, or it’s the wrong colour or the wrong style. If we haven’t got it, we wish we did, or if we have lost it we try to make it look as though we still have it. We worry about the ones that fall out, and the ones that turn grey, or worse yet, white! And then there are those who worry about whether hair is dead or alive. ‘Living things grow, OK? Hair grows, OK? So hair must be alive, OK?’ ‘No,’ came the answer, ‘hair’s not alive, it’s dead!’ ‘Well,’ I said, ‘how does it grow, then, if it’s dead?’ (Hackett, 1984) The short answer to this paradox is that the hair root embedded in the skin is a living tissue and grows to produce the hair we see, which is a dead tissue. It is ‘as dead as rope’ (Montagna, 1963), in spite of what some of the advertising of the cosmetic and hair care companies would try to convince us. The living part, the hair follicle, is an appendage of the skin and develops as an invagination of the epidermis. It is a dynamic and complex organ of considerable interest to molecular biologists as a model for cell differentiation, tissue development, and gene expression and control (Hardy, 1992). The natural growth process of the follicle causes hairs to be shed on a periodic basis, but they are replaced and this cycle is repeated over and over again, although the replacement hair may well be different from its predecessor. It is this periodic shedding that causes hair from a person’s body to be commonly found on clothing and at crime scenes, and which makes them so useful as associative evidence in forensic science. In comparison to many mammals humans are quite glabrous, sometimes being referred to as ‘the naked ape’. While the number of follicles in humans may be less than in many of the other mammals, we do, however, have as many hair follicles as other primates, and the generally hairless appearance results from the fact that many of the follicles have become so small that the hairs they produce are not easily visible on the surface of the skin. The only truly naked mammals are the whales, which have lost all their follicles (Montagna, 1963). 1 Forensic Examination of Hair So what do humans have hair for, especially since there are only a few areas on the body of highly visible growth? It would seem that some is ornamental—perhaps the same as the mane on the lion (Montagna, 1963). Scalp hair undoubtedly comes into this category, and it also has a protective function in shielding the scalp from the sun. The eyebrows and eyelashes, which like scalp hair are present from birth, probably also have protective roles, serving to keep the eyeballs free from dust, rain and sweat (Montagna and Parakkal, 1974; Robbins, 1988). Hairs in other areas may have special functions; those of the outer ear and those of the anogenital regions, for example, provide barriers to the entry of foreign matter such as dirt and insects to these body orifices. The hairs in the nostrils slow down the incoming air and so provide some temperature control, as well as trapping insects and dust and the like. The fact that the hairs at the other main areas of growth (male beard region, pubic region and axillary region) do not fully develop until sexual maturity might suggest that they have an erogenous role (Montagna and Parakkal, 1974). However, the most important role of hairs in humans may be that of sensory receptors. All hair follicles, and particularly those of the human face and anogenital region, are surrounded by sensory nerves, and these nerves react to pressure on the hair shaft, making them very sensitive to touching (Montagna and Parakkal, 1974). Hair growth and appearance are under genetic control (Kaszynski, 1987). Hairs originate directly from the person and may be subjected to treatments (such as cutting method and dyeing) which may further aid in forensic comparison. But it must be remembered that each hair is an individual that grows independently of the others, and no two hairs will be exactly alike in all details. To make a reasoned and reasonable forensic examination and comparison of human hair it is therefore necessary to have an understanding of how hairs develop and grow, and of some of the factors which cause variability in these processes, both within individuals and between individuals. It is the aim of this chapter to provide some of that information. 1.2 Embryology of Human Hairs 1.2.1 Development of the Hair Follicle Hair follicles develop in utero as a downgrowth or invagination of the epidermis into the dermis. Although the development is a continuous process, it is convenient to describe it as a series of stages, namely, pre-germ, hair germ, hair peg, bulbous peg and finally, the hair follicle. These stages have been well described in detail by Pinkus (1958) and are illustrated in Figure 1.1. Other authors use eight stages (Chapman, 1986). The Pre-germ Stage The first sign of follicle development occurs at about eight weeks after conception as a local clustering of cells in the basal layer of the epidermis. The bulge so formed on the underside of the epidermis is called the pre-germ (Pinkus, 1958) or primitive hair germ (Montagna and Parakkal, 1974). Numerous mitoses occur in the immediate surroundings of the hair germ. The Hair Germ Stage The second stage follows quickly, and by about eleven weeks after conception the epidermal basal cells elongate perpendicularly to the skin, producing on one side a bulge in the external surface of the epidermis and growing down on the other side into the dermis. Additional cells start to accumulate above the basal layer but beneath the periderm of the skin above the developing follicle (Pinkus, 1958), and at the same time, fibroblasts and mesenchymal cells increase in number below the hair germ to form the beginnings of the dermal papilla (Ebling, 1980). The basement membrane starts to appear at this stage (Pinkus, 1958) and melanocytes (pigment cells) can be seen scattered throughout the epidermis and the developing hair germ (Mishima and Widlan, 1966; Ortonne and 2  Physiology and Growth of Human Hair Figure 1.1 Schematic diagram of the development of the hair follicle in the foetus. Source Ebling (1980); used with permission Thivolet, 1981; Holbrook et al., 1989). Nerves are prominent in the mesenchyme associated with the developing follicle (Holbrook et al., 1993). The hair germ is initially asymmetric, with one side forming a right angle with the epidermis, the other side slanting and merging gradually with the basal layer. Continued downward growth of the germ is oblique, however, and eventually the column of cells takes up the slanting position of the fully formed follicle (Pinkus, 1958). The Hair Peg Stage Continued cell division causes the hair germ to grow obliquely down into the dermis as a solid column (or peg) of epithelial cells, the outer layer being continuous with the basal epidermal layer (Holbrook et al., 1993). The cells at the leading edge of the peg are longitudinal and appear to push the cluster of fibroblasts (the developing dermal papilla) further down into the dermis ahead of them. A sheath of mesodermal cells, contiguous with those of the papilla, surrounds the entire peg. Cell division continues 3 Forensic Examination of Hair and glycogen appears in the cells, firstly in those of the central core of the developing follicle, and later in the peripheral basal cells (Pinkus, 1958; Montagna and Parakkal, 1974). Melanocytes are distributed almost randomly throughout the peripheral and inner cell layers of the follicle (Mishima and Widlan, 1966). The Bulbous Peg Stage Tissue differentiation begins at this stage (Pinkus, 1958). The advancing end of the developing follicle broadens and then becomes concave, eventually enclosing the dermal papilla inside its hollow bulb of germinative cells. The papilla remains connected, however, to the dermis via a basal stalk through an opening in the base of the follicle bulb. The melanocytes become localized in the peripheral layer of the outer root sheath above the level of the widest part of the dermal papilla, and also among the germinative cells above the dermal papilla and in the lower part of the bulb (Pinkus, 1958; Mishima and Widlan, 1966; Ortonne and Thivolet, 1981; Holbrook, 1991). At this stage two epithelial swellings or outgrowths appear on the posterior (that is, under the slant) side of the developing follicle. The lower, larger one remains solid, its cells becoming rich in glycogen, together with the rest of the cells of the follicular peg. This swelling is called the bulge, and eventually becomes the site of attachment for the arrector pili muscle. The bulge also marks the point in the follicle above which all follicular components persist throughout the subsequent hair cycles, whereas the parts below are resorbed during the catagen phases (Pinkus, 1958; Montagna and Parakkal, 1974), and it has recently been demonstrated that this region is the location of the pluripotent stem cells that produce the elongation of the follicle in its anagen (growing) phase (Lavker et al., 1993; Kim et al., 1996). The higher swelling is more rounded; its cells accumulate lipid and form the sebaceous gland. The central cells in the peg elongate backwards towards the epidermis, defining what will become the hair canal, but the developing follicle remains a solid mass of cells and there is no actual canal or channel in the follicle at this stage nor is there an orifice in the skin. At this point mesodermal cells near the sebaceous gland form into a slender row parallel to the posterior side of the follicle. This row of cells forms the arrector pili muscle (also called the erector muscle), which gradually extends downwards to the bulge (Pinkus, 1958). An apocrine gland now forms in some follicles from a small knob of cells on the posterior side of the follicle above the level of the sebaceous gland duct. The knob grows into a solid cord with a pointed tip and irregular shape (Pinkus, 1958). The germinative cells in the hair bulb that are adjacent to the dermal papilla now begin to divide actively. The inner root sheath cells differentiate first, aligning themselves along the follicle and forming the so-called hair cone above the dermal papilla. The hair cone elongates, pushed up by dividing cells from below, forcing its way upwards between the cells of the central core of the hair canal without causing any cell death in this area (Breathnach and Robins, 1981). When the tip of the hair cone is about halfway up the follicle its outer layer hardens and the central cells inside it start to differentiate to form the tip of the new hair. By the time the hair cone reaches the level of the sebaceous gland, the tip of the hair starts to keratinize and the hair cone fragments (Chapman, 1986). The inner root sheath thus does not extend above the level of the sebaceous gland opening (Pinkus, 1958; Chase and Silver, 1969). The new hair fibre finally breaks through the epidermis and appears above the skin at around 19–21 weeks. This lanugo (foetal) hair is extremely fine with no medulla and the tip is free of pigment (Pinkus, 1958) (see section 1.7.1). The Hair Follicle It is clear from their origin that hair follicles are an epidermal tissue. The cells of the basal layer of the epidermis which originally extended down into the dermis as the hair peg become the outer root sheath of the follicle (Chase and Silver, 1969), whereas the cells in the follicle core and which form the hair proper are continuous with the intermediate cell layer of the epidermis (Holbrook et al., 1989). (The papilla always remains part of the dermis.) The features of the fully developed hair follicle are described in detail in section 1.3. 4  Physiology and Growth of Human Hair 1.2.2 Initiation and Growth—Chronology Follicle development is initiated in the foetus at different times depending on the region. Follicle downgrowths are seen first between the second and third months of development and the activity at this stage occurs mainly on the upper lip and eyebrow region, with some on the chin (Pinkus, 1958). General hair development then begins, starting with the forehead and scalp, at about the fourth foetal month and continues through the fifth month (Szabo, 1958; Montagna and Parakkal, 1974). As the skin of the foetus expands with growth, new follicles form between the original primary ones. The new follicles consist of both primary and secondary (i.e. smaller) types, and they occur initially in groups of three (Montagna and Parakkal, 1974), although in many regions only one of them will persist and go on to form a hair (Holbrook, 1991). Sebaceous glands develop at about the sixth month (Butcher, 1950). The lanugo hairs that are formed initially grow at the rate of 2 mm per week (Baum et al., 1974), but this slows down (catagen phase) and eventually stops (telogen stage) as their follicles go through their first hair growth cycle. The lanugo hairs are shed in utero, beginning with the face and head, between the seventh and eighth months. They are replaced by new lanugo hairs or by vellus hairs (depending on the region) growing from the same follicles. This change continues up to birth or even into the first few months of post-natal life (Butcher, 1950). Thus at birth, follicles will be in all stages of the hair cycle (Montagna and Parakkal, 1974). New follicles do not develop to any great extent after birth (Pinkus, 1958), and with the further expansion of the skin the foetal follicle distribution pattern tends to be lost, creating the more random arrangement of hairs usually seen for human scalp hair (Ellis, 1958). Thus the follicles formed in the foetus provide the hairs for the foetus, the child and the adult. 1.3 Structure of the Hair Follicle The hair follicle is a dynamic organ in which division, differentiation and migration of cells occur in the various tissues of which it is composed. These processes give rise to the growth of the hair fibre, which is formed as the result of the biosynthesis and hardening of the contents of the medulla, cortex and cuticle cells of the hair shaft. This growth, however, is not a continuous process, but one which ceases periodically and then starts again, to be repeated in what is known as the hair growth cycle (Chase, 1954, 1965). The stage of this cycle in which the hair is growing actively is called anagen, when the follicle is full size and maximally biochemically active. This is followed by a quiescent or regression phase (catagen) when metabolic activity (and hair growth) slows down and eventually stops (the resting phase, telogen) (Dry, 1926). This process is discussed in more detail in the context of hair growth in section 1.6. The main cellular features of the mature, growing hair follicle are illustrated in Figure 1.2. A section of a growing follicle from human scalp is shown in Figure 1.3. The hair follicle consists of several approximately cylindrical and concentric cell layers. The outer root sheath (ORS), the most external component, encases the other cell layers of the follicle and is contiguous with the epidermis. The inner root sheath (IRS) is composed of three distinct layers, the Henle, Huxley and IRS cuticle layers in that order, which encase the growing hair fibre. The mature fibre contains at least two cell types, the surface layer or cuticle consisting of flattened overlapping cells and the main central cortex of spindle-shaped cortical cells. The hair may include a medulla, a core of condensed cells of a third type stacked in the centre of the cortex. Except for the ORS, the cells of these different layers derive from the germinative cells which reside in the follicle bulb and proliferate during the anagen (growth) phase of the hair cycle. Specialized mesenchymal cells form the dermal papilla which protrudes into the base of the follicle bulb but is separated from the germinative cells by a basement membrane. The basement membrane covers the interfaces of the epidermis and follicles. External to it and around the follicle is the glassy layer consisting of orthogonally arranged collagen fibrils (Rogers, 1957). External to this again is the dermal sheath, a connective tissue layer of a collagen network and fibroblasts. 5 Forensic Examination of Hair Figure 1.2 Schematic diagram of the pilosebaceous unit showing various main features and the regions where the main events of cell proliferation and keratinization take place. The cells in the bulb region move into different streams that give rise to the cortex, the cuticle, the medulla and the inner root sheath. Modified from ‘The role of keratin proteins and their genes in the growth, structure and properties of hair’ by B.C.Powell and G.E.Rogers, in Formation and Structure of Human Hair, edited by P.Jolles, H.Zahn and H.Hocker, © 1997 Birkhauser Verlag Basel/ Switzerland Attached to the follicle is the arrector pili muscle and, to some follicles, one or more sebaceous glands. The combination of follicle and sebaceous gland is known as the pilosebaceous unit (Pinkus, 1958; Chase and Silver, 1969). Networks of nerves and of blood capillaries (not shown in Figure 1.2) also surround the follicle. The size of a follicle depends on a number of factors, including the region of the body and whether it is a primary or secondary (smaller) follicle (both of which relate to the type of hair it 6  Physiology and Growth of Human Hair Figure 1.3 Longitudinal section through a growing hair follicle from human scalp, showing various cellular components. Stained with haematoxylin/eosin. Note that shrinkage during the section preparation has caused some separation of layers. Co, cortex; Cu, cuticle; DP, dermal papilla; Ge, germinative cells; IRS, inner root sheath; K, keratogenous zone; M, medulla; Me, melanocytes; ORS, outer root sheath. Bar = 100 µm produces), and to the stage of the hair cycle. The models developed by Montagna and Van Scott (1958) well illustrate the different sizes and types. A mature anagen follicle of a terminal hair is about 4–5 mm in length (Kligman, 1962). In structural terms the region of the follicle below the bulge (the attachment point of the arrector pili muscle) is called the lower follicle. This is also known as the transient zone, as it is the part of the follicle which regresses during the catagen phase of the hair cycle. The section between the bulge and the sebaceous gland duct is referred to as the isthmus, and the infundibulum is the region from the isthmus to the epidermis. Thus the isthmus and the infundibulum together make up the permanent zone of the follicle (Figure 1.2) which persists through all stages of the hair cycle. The follicle is functionally divided into four zones, also shown in Figure 1.2. These zones are based on cellular and biochemical activity and are helpful to aid description of the follicle components and their differentiation. The zones are (1) the cell proliferation and differentiation zone at the base of the bulb, (2) the keratin gene expression zone in the upper bulb, (3) the keratogenous zone in which hardening of the fibre occurs, and (4) the zone of IRS degradation. The structure and development of various components of the pilosebaceous unit are described in more detail below. 7 Forensic Examination of Hair 1.3.1 The Hair Bulb The area of active cell division in the follicle is the lower bulb, mostly in the part below what was called the critical level by Auber (1952) (and which is an imaginary line drawn across the follicle at the level of the widest part of the dermal papilla), and also in the area adjacent to and covering the apex or dome of the dermal papilla (Epstein and Maibach, 1969). The epithelial cells of this dividing cell region are undifferentiated and are characterized by a high nuclear to cytoplasmic ratio. The cytoplasm contains a few mitochondria and smooth membranes but no morphologically identifiable filaments (Birbeck and Mercer, 1957a). These cells are now referred to as ‘germinative’ cells and not ‘matrix’ cells as in the past. This not only is a better description of their function but also avoids confusion with the customary use of the term ‘matrix’ to describe the complex of proteins that becomes associated with the keratin intermediate filaments during keratinization. The germinative cells are several layers deep and are organized so that cell division gives rise to the concentric layers of the follicle and the hair fibre. Up to six distinct cell streams may exist (Figures 1.2 and 1.3) and by continuous division the cells move outwards and upwards to form the tissues of the follicle and the hair fibre. The most central cells give rise to the medulla (when it is present), then, progressing outwards, the next layers respectively give rise to the hair cortex, the hair cuticle, the cuticle of the IRS, the Huxley layer of the IRS and, at the periphery, the Henle layer of the IRS. Differentiation and development in each of the cell streams are distinct from those in adjacent streams (Birbeck and Mercer, 1957a). It has been estimated that no more than 20 per cent of the bulb cells differentiate into the fibre cortex, the rest forming the IRS layers (Wilson and Short, 1979). Melanocytes (pigment-producing cells) are present among the germinative cells, mostly attached to the basement membrane around the apex of the dermal papilla (Birbeck and Mercer, 1957a; Swift, 1977; Tobin et al., 1995) but some are also found in the ORS (Montagna and Parakkal, 1974; Takada et al., 1992; Tobin et al., 1995). Each melanocyte has a number of dendrites (processes) which extend upwards between the presumptive cortical cells (Montagna and Parakkal, 1974; Swift, 1977). The germinative cells of the hair bulb are among the most actively dividing cells in the whole body. Cell renewal times reported have ranged from 8 hours (Bullough and Laurence, 1958) to 23– 72 hours (Van Scott et al., 1963), but they are difficult to measure and these figures should be taken only as estimates (Epstein and Maibach, 1969). The extensive cell proliferation requires adequate supplies of oxygen and metabolites, such as glucose, fructose and pyruvate, and these are obtained via the blood capillaries (Bullough and Laurence, 1958). Energy for the synthetic activities is provided in the follicle cells through the Embden- Meyerhof and tricarboxylic acid cycles (Adachi and Uno, 1969; Kealey et al., 1991). When follicles become active in the anagen phase the rate of ATP synthesis is doubled as a result of a four-fold increase of activity of the pentose cycle, producing not only increased energy but also essential substances required for the fatty acid and nucleic acid metabolism of the follicles (Adachi and Uno, 1969). Phosphoglucomutase (PGM), an enzyme which catalyzes one of the steps of the breakdown of glycogen to glucose for use in the metabolic cycles, is present in the growing hair root in sufficient quantities to enable the easy detection of its polymorphic forms (Twibell and Whitehead, 1978; Whitehead et al., 1981). The availability of adequate supplies of essential amino acids is vital for normal hair growth. A deficiency in the supply of sulphur amino acids results in lower cell division rates in the follicle germinative cells, a lowered rate of hair growth and a change in the pattern of keratin synthesis (Fratini et al., 1994). The high mitotic rate of the germinative cells in the growing bulb leaves these hair roots susceptible to anti-mitotic agents used for anticancer therapy. Treatment with these drugs causes the anagen roots to atrophy, and the hairs fall out. This can be quite dramatic (and traumatic), since about 90 per cent of follicles in the scalp are in anagen at any one time (Orentreich, 1969). There is a highly significant relationship between the hair root diameter and the amount of DNA in the root (Bradfield and Gray, 1975). A single anagen root can yield about 50 ng (von Beroldingen et al., 1989). The genomic DNA in the germinative cells of the bulb can be extracted more easily than that in the differentiated cells further up the follicle and in the hair shaft, and hair roots are thus amenable to DNA typing in a variety of systems following polymerase chain reaction amplification (Higuchi et al., 1988; Westwood and Werrett, 1990; Fregeau and Fourney, 1993). 8  Physiology and Growth of Human Hair 1.3.2 Medulla Cells Most of what is known about the differentiation of medullary cells comes from studies of animals other than humans, but the essential stages are similar, regardless of species. Medullary cells arise from the germinative cells of the bulb adjacent to the apex of the dermal papilla (Rogers, 1964). Changes can be seen in this cell stream before they are detectable in the other cell types. At a level about 3–4 cells above the papilla, obvious differentiation into medulla cells begins with the appearance of characteristic electron-dense, amorphous granules. These granules vary in size and are not bound by membrane (Parakkal and Matoltsy, 1964). They contain the protein trichohyalin and stain strongly for arginine (Rogers, 1963). The number and size of the granules increase at later stages of development and large amounts of glycogen are seen in the cells. The mitochondria swell and vacuolate and vesicles appear in the cytoplasm as the cells move higher in the follicle. The nuclei degenerate and the granules tend to become irregular and coalesce. In a sudden transformation, from one cell to the one above, the granules fuse, giving rise to hardened protein showing a generally granular substructure and which contains citrulline (Rogers, 1963; Harding and Rogers, 1971; Rogers et al., 1977). The hardened protein is not overtly filamentous (Rogers, 1964). It is, however, highly insoluble due to the formation of e-(?-glutamyl)lysine crosslinks (Harding and Rogers, 1972a, 1972b; Rogers and Harding, 1976). When the granules fuse, large intercellular spaces form and the cell contents tend to coalesce at the cell periphery (Parakkal and Matoltsy, 1964). In the mature hair the medulla cells form a central core surrounded by cortical cells, although the cortical cells may interrupt the core on occasion if there has been a pause in the production of medullary cells (see section 1.4.1). 1.3.3 Cortical Cells The presumptive cortical cells are derived from the central cells in the bulb, surrounding those which form the medulla. Their differentiation and development in the human follicle have been described in detail by Birbeck and Mercer (1957a). The cells initially contain large, roughly spherical nuclei and long, thin mitochondria. The characteristic feature of the cytoplasm is numerous clusters of small dense particles of about 20 nm in diameter. Filamentous material is not seen in the cells at this early stage. The cells are rounded at first but the surfaces become corrugated, with only limited contacts between the cells. Intercellular gaps of up to 2–3 µm are common, and these persist as the cells move past the melanocytes around the upper part and apex of the dermal papilla in the mid to upper level of the bulb. The gaps allow the dendrites of the melanocytes to penetrate between the developing cortical cells. The melanin pigments synthesized in the melanocytes are packaged into membrane-bound granules which move out along the dendrites. It is believed that the melanin-laden dendrites are engulfed by the membrane of a recipient cortical cell, pinched off and drawn into the cytoplasm in a phagocytic process (Birbeck and Mercer, 1957a; Swift, 1977). Partial digestion of these pieces releases the pigment granules inside the cortical cells. As the cells move to the upper level of the bulb they rapidly elongate into a spindle shape and the cell contents, including the nucleus and mitochondria, become oriented parallel to the axis of the follicle. Filaments are first seen in the cells at this stage; they are also aligned with the axis of the cell. This is a period of protein synthesis, but the keratin produced is maintained in the sulphydryl state (Rogers, 1959a). As the cells move through the neck of the bulb the bundles of filaments rapidly increase in length and width to form macrofilaments (also called macrofibrils), with the pigment granules lying in rows between them. Further up the follicle in the keratogenous zone, the macrofibrils become more clearly defined and more filaments (microfibrils), together with amorphous interfilamentous material, are seen. The macrofibrils increase in size and length to fill eventually almost the whole cell, trapping the nucleus and cellular remnants at their interces (Birbeck and Mercer, 1957a). In this process the nucleus becomes 9 Forensic Examination of Hair considerably distorted, being converted into roughly a spindle shape like the cells, and located in the centre of the cell surrounded by macrofilaments (Swift, 1977; Rook and Dawber, 1982). The arrangement and composition of the macrofibrils and the keratin microfibrils, which are now called keratin intermediate filaments (keratin IF), are discussed in more detail in section 1.4.2. Finally, at the upper level of the keratogenous zone, in a conversion extending over the length of several cells, the cell contents no longer react for sulphydryl groups. This change in reactivity occurs as the result of almost complete disulphide bond cross-linking of the proteins in the last step of keratinization. During the terminal stages of differentiation the cortical cells harden and dehydrate, reducing the hair fibre diameter by about 25 per cent (Rook and Dawber, 1982). A continuous layer of intercellular material, referred to as the cell membrane complex (CMC), appears between the cortical and cuticle cells and at the junctions between cortex and cuticle (Birbeck and Mercer, 1957a, 1957b; Rogers, 1964). From this point on the tissue of the hair fibre is biologically inactive and is considered ‘dead’. 1.3.4 Cuticle Cells Cuticle cells arise from a single layer of germinative tissues outside those that form the cortex (Birbeck and Mercer, 1957b). The first sign of their differentiation occurs at a level between the lower and mid- bulb as a smoothing-out of the cell membrane. The smoothing results from the formation of small areas of contact between successive presumptive cuticle cells. As differentiation proceeds, the number and area of these cell contacts increases until they completely spread over the surfaces between the cuticle cells. This cementing converts the germinative cells into an organized layer of cuboidal cells at about mid-bulb level (Birbeck and Mercer, 1957b). The cells then undergo a slow further change of shape as they move up the bulb and through the neck region, in which they are elongated and flattened, and then tilted to slide over each other to produce a layer of overlapping cells (Swift, 1977). As the cells tilt, their nuclei move to the lower end of the cell and are squashed (Birbeck and Mercer, 1957b). Granules of about 30–40 nm diameter are initially seen around the periphery of the cells, and these grow in size on the outer side of the cell until granular material fills this half of the cell, compressing the nucleus and cytoplasm into the other half. The granular material rapidly transforms to produce the exocuticle and the ‘A’ layer of the mature fibre cuticle, and the underlying flattened zone of residual cytoplasm condenses and dehydrates to form the endocuticle (Swift, 1977). These aspects are discussed further in section 1.4.3. 1.3.5 Inner Root Sheath The inner root sheath (IRS) tissue comprises three layers of cells which arise as separate but adjacent concentric cylindrical layers from the germinative cells of the hair bulb (Figure 1.2). The layers are each only one cell thick (Birbeck and Mercer, 1957c), but combined they account for about 80 per cent of the cells emerging from the hair bulb (Wilson and Short, 1979; Rogers et al., 1989). The IRS layers are on the outside of the layer that forms the hair fibre cuticle, and indeed the first layer of the IRS is called the IRS cuticle. Outwards from this is the Huxley layer, and then the Henle layer (which is adjacent to the outer root sheath (ORS)). During hair growth the IRS moves up the follicle inside the surrounding ORS as a result of the division of the presumptive IRS germinative cells. Maturation and hardening of the IRS cells occurs low down in the follicle, with the result that the IRS moulds and shapes the forming hair (Straile, 1965; Kassenbeck, 1981). The upward movement of the IRS cells is more rapid than that of the precortical cells of the hair (Epstein and Maibach, 1969), so that the IRS forms a sleeve that physically supports the forming hair fibre. The support of the hair shaft continues up to the level of the sebaceous gland duct, at which point the IRS cells dissociate and fragment, presumably as the result of proteolytic enzyme activity (Straile, 1965; Fraser et al., 1972), and slough off into the pilary canal (Straile, 1965), releasing the emerging hair (Figure 1.2). The IRS also provides the mechanical function of holding the growing hair in the follicle. This is achieved by an interlocking interaction of the IRS cuticle cells, 10  Physiology and Growth of Human Hair which form as scales that point downwards, with the similar but opposing scales on the cuticle of the hair fibre (Straile, 1965). Thus a quick tug on a growing hair can remove the hair with the IRS attached to its root. Cellular differentiation events are similar in all three layers of the IRS cells but totally different from those that occur in the fibre cortex. The first stage of differentiation is the appearance of amorphous granules of trichohyalin protein (Birbeck and Mercer, 1957c; Rogers, 1964) that is rich in arginine (Rogers, 1963). At the same time, filaments of about 7 nm diameter are found in the cytoplasm (Rogers, 1964). These differentiation processes occur at different rates in each IRS layer (Birbeck and Mercer, 1957c; Fraser et al., 1972; Swift, 1977). In the Henle layer the changes begin immediately above the germinative cells. Similar granules and filaments subsequently appear in the cells of the IRS cuticle and then the Huxley layer (Birbeck and Mercer, 1957c). As the IRS cells move up the follicle the number and size of the granules increase, as does the number of filaments. The final stage of differentiation is a sudden and dramatic transformation, occurring from one cell to the next (Birbeck and Mercer, 1957c; Rogers, 1963, 1964). Granules are no longer visible, and the cells are filled with filaments oriented in the direction of fibre growth (Fraser et al., 1972; Rogers et al., 1991). The cell contents now stain strongly for the amino acid citrulline (Rogers, 1963; Rogers and Harding, 1976). The granule-to-filament transformation occurs at different levels in the three layers of the IRS; the outermost Henle layer hardens first, at about the level of the top of the bulb, whereas the Huxley layer converts last, at approximately the same level as the final events of the hair fibre formation. The trichohyalin molecule and its gene were first characterized by Fietz et al. (1993) in the wool follicle. The sheep protein is 1549 amino acids long and has a molecular weight of 201,172. It is hydrophilic, and is unusual in that it has one of the highest contents (over 57 per cent) of charged amino acids of any protein. It is further characterized by the four amino acids, glutamic acid, glutamine, arginine, and leucine, constituting over 75 per cent of the protein. Human trichohyalin has also been characterized, and is a protein of 1897 amino acids which is homologous with the sheep protein (Lee et al., 1993b). It is expressed from a single copy gene that maps to chromosome 1q21.1–21.3 (Fietz et al., 1992; Lee et al., 1993b). At the time of transformation and hardening, up to 90 per cent of the arginine residues of trichohyalin are converted to citrulline by the action of a peptidylarginine deiminase (PAD) enzyme (Rogers and Harding, 1975, 1976; Rogers et al., 1977). The formation of uncharged citrulline residues from positively-charged arginine residues causes a change in ionic charge of the protein and results in the protein undergoing a conformational change. It is postulated that the protein disperses as a matrix between the filaments (Fietz et al., 1993). The protein is then insolubilized by the formation of e-(?-glutamyl)lysine cross-links by follicle transglutaminase (Harding and Rogers, 1972b; Rogers and Harding, 1976; Rothnagel and Rogers, 1986; Rogers et al., 1989). The molecular biology of trichohyalin is discussed further in section 1.5.1. At one time it was believed that in the transformation of the cells the trichohyalin in the granules gave rise to the filaments (Birbeck and Mercer, 1957c; Rogers, 1964), but it is now accepted that the trichohyalin forms a matrix between the intermediate-type filaments (Rothnagel and Rogers, 1986; O’Guin et al., 1992; Fietz et al., 1993; Lee et al., 1993a). This enmeshing of trichohyalin with separately formed intermediate-type filaments in the IRS is an essential difference between the IRS and the medulla, in which the trichohyalin granules fuse together to form a solid mass (Lee et al., 1993a). Definitive identification of the intermediate filaments in the mature IRS cells has yet to be made, but there is some evidence that they do contain keratin IF proteins (Stark et al., 1990). 1.3.6 Outer Root Sheath The outermost layer of the follicle is the outer root sheath (ORS). Unlike the other layers, the ORS has its own autonomous cell population. The sheath is a single layer at the base of the bulb, but higher up the follicle the number of layers increases. The first increase is to two layers, with the cells on the outer side being elongated and those on the inner layer being flattened (Rook and Dawber, 1982). The ORS cells are attached to each other, and to the cells of the Henle layer of the IRS, by desmosomes (Swift, 1977). At the level of the neck of the bulb the number of layers increases even more (Rook and 11 Forensic Examination of Hair Dawber, 1982), so that by the time the cells reach the level of the sebaceous gland the ORS layers are continuous with the various layers of the epidermis (Swift, 1977). Fibrils, and granules which resemble the keratohyalin granules of the epidermis, form in the cells high up in the follicle. Epidermal keratins, rather than hair type IFs, are present in the ORS (Coulombe et al., 1989). It has now been demonstrated that the stem cells which give rise to the follicular germinative cells of the hair bulb at the beginning of each anagen reside in the bulge of the ORS, the swelling located just below the sebaceous gland (Lavker et al., 1993; Kim et al., 1996). The inner layer of cells of the ORS that lies next to the Henle layer of the IRS is known as the ‘companion cell layer’ (Orwin, 1971). These ORS cells are elongated and flattened, and originate in the bulb near the dermal papilla as a cell type which is distinct from the other cell types in the ORS. They are the first of all types of cells to accumulate keratin-like filaments, and in these cells the filaments have a circumferential orientation. The companion layer cells can be as thin as 1 µm and they are tightly apposed to the hardened cells of the Henle layer of the IRS rather than to the other ORS cells (Rogers, 1964). This has led to the suggestion that the companion cells of the ORS assist in the relative movement of the IRS and the ORS by moving towards the surface of the skin as part of the fibre-IRS complex (Rogers, 1964; Orwin, 1971). 1.3.7 Dermal Papilla At the base of the follicle and enclosed by the germinative cells of the (anagen) hair bulb is the dermal papilla, a roughly egg-shaped tissue that is derived from fibroblasts in the dermis at the earliest stages of development of the follicle in the foetus. The fibroblasts aggregate ahead of the epithelial plug of the developing follicle as it grows down into the epidermis (Oliver and Jahoda, 1989). During the development of the follicle the developing papilla interacts with the ectoderm to specify the location, timing and type of follicles to be formed (Oliver and Jahoda, 1981). The papilla is the only dermal component of the follicle; in growing follicles it is attached to a basal plate of connective tissue by a narrow stalk. Although it is eventually surrounded by the germinative cells of the growing bulb, it is separated from them by a basement membrane (basal lamina) (Montagna and Parakkal, 1974). The dermal papilla is essential for hair growth and is responsible for the control of the hair cycle (Oliver, 1971), but the nature of the signals and signal pathways is not yet known. Removal of the dermal papilla stops hair growth, but after a short lag period a new papilla is generated and hairs of normal length are grown and continue to grow (Oliver, 1971; Oliver and Jahoda, 1981, 1989). Hair growth can also be restored by implantation of a dermal papilla (Cohen, 1965; Oliver, 1971; Oliver and Jahoda, 1981). When the dermal papilla, the hair bulb and up to the lower one-third of the follicle are removed, new smaller papillae regenerate and hairs shorter than normal will grow (Oliver and Jahoda, 1981). It was believed that permanent hair removal by electrolysis or electrodesiccation was achieved when the papillae only were destroyed (Van Scott and Ekel, 1958), but Oliver (1971) and Oliver and Jahoda (1981) have demonstrated that more than the lower third of the follicle must be destroyed as well if the dermal papillae are not to regenerate and stimulate renewed hair growth. The explanation for this lies in the recent discovery that the stem cells that give rise to the germinative cells of the follicle bulb reside in the middle third of the human hair follicle (Kim et al., 1996). The dermal papilla is the only permanent part of the lower follicle, remaining through the hair cycle while the hair-producing germinative cells disappear and reappear. It does nevertheless have a cycle of its own, in which changes to its structure and synthetic activities occur in phase with the hair cycle (Oliver and Jahoda, 1981). During catagen the papillary cells lose cytoplasmic volume to the point where they contain virtually only a nucleus. The basal lamina shrinks and crinkles and the dermal papilla becomes detached from the bulb (Montagna and Parakkal, 1974). The mucopolysaccharides and alkaline phosphatases that are present for synthetic activity in anagen decrease, and are absent in telogen (Oliver, 1971). The dermal papilla, however, remains as a condensed ball of cells at the base of the shortened follicle throughout telogen. It is believed that the papilla provides the 12  Physiology and Growth of Human Hair stimulus for cell proliferation in the hair germ at the initiation of proanagen and thus the hair cycle may in fact be dependent on a papillary cycle (Oliver and Jahoda, 1981). As well as controlling hair growth, the dermal papilla also controls the physical characteristics of the hair. Van Scott and Ekel (1958) have shown that there is a direct correlation between hair size and dermal papilla size, and between mitotic activity of the germinative cells of the bulb and the number of cells in the papilla. Hair follicles that grow large (diameter) hairs have large dermal papillae, while smaller follicles with smaller dermal papillae produce thinner hairs (as seen for example in the case of alopecic follicles in the balding scalp). In normal scalp the average volume of the germinative cells of the hair bulb is about ten times the volume of the papilla, and the ratio of the number of mitotic germinative cells in the bulb to the number of cells in the dermal papilla is about 1 : 9 (Van Scott and Ekel, 1958). Hairs with multiple medullae have multiple apices (one for each medulla) to their dermal papillae (Montagna and Parakkal, 1974). Changes in the shape of the upper surface of the dermal papilla during the growth of a hair can, in conjunction with the inner root sheath, mould the adjacent layers of migrating medulla, cortex and cuticle cells, thus producing a hair of changeable contour (Straile, 1965). 1.3.8 Sebaceous Glands Sebaceous glands in humans are generally associated with hair follicles, the combination being known as the pilosebaceous unit (Chase and Silver, 1969). The glands are found everywhere on the human skin except the palms, soles and dorsum of the feet (Strauss et al., 1991), and are particularly numerous on the face and scalp (Montagna and Parakkal, 1974). Sebaceous glands form in utero as an outgrowth of the follicle, the beginning of their development being visible at the bulbous peg stage (Pinkus, 1958). The glands are well formed in the foetus, but after birth they reduce in size and enlarge again at the onset of puberty (Strauss et al., 1991). Human sebaceous glands consist of multiple aggregates of acini (lobes) which attach to a common duct. The cells within the glands convert their glycogen into lipid within the cellular membrane systems (Holbrook, 1991) and eventually fragment to form sebum (Montagna and Parakkal, 1974). The glands in humans are often attached to the follicular canal by a short duct at a level above the bulge, and the sebum is discharged through this duct and on to the skin via the canal. However, in some regions of the skin the sebaceous glands are not connected to the follicles, and open directly at the skin surface (Montagna and Parakkal, 1974). Thus it would seem that sebum is a phenomenon of the skin and is not essential for hair growth. The main components of human sebum are squalene, wax esters and triglycerides, and it has been proposed that its function is to waterproof the hair, but this would not be a significant factor in humans (Strauss et al., 1991). 1.3.9 Blood Supply Hair follicles are well supplied with blood through a network of capillaries and arterioles (Montagna and Parakkal, 1974; Sato et al., 1976). These vascular systems, which form a continuous unit for each follicle, are branches of the dermal plexus or the musculocutaneous arteries (Montagna and Parakkal, 1974). In an active follicle, the capillaries surround the lower third from the base to just above the bulb in a latticework pattern of parallel vessels connected by cross-shunts. There are fewer cross-shunts further up the follicle in the region from the top of the bulb to the level of the sebaceous gland, but a dense network is seen once again around the gland and the pilary canal. Loops of capillaries also form a vascular ring around the orifice of the growing follicle under the epidermis (Montagna and Ellis, 1957). In large terminal hair follicles the dermal papilla is penetrated in its centre by a large tuft of capillaries, some of which extend to the wall of the inner side of the matrix of the follicle (Montagna and Ellis, 1957). The larger the dermal papilla, the more capillaries it contains (Ryder, 1956). Smaller follicles 13 Forensic Examination of Hair with smaller dermal papillae, such as those of vellus hairs and hairs in alopecic skin, are surrounded by a much simpler system of capillaries (Allegra and De Panfilis, 1981), with only a few around the lower part of the bulb and none entering the dermal papilla (Montagna and Ellis, 1957). During catagen as the follicle bulb atrophies, it moves upwards in the dermis and partially out of the network of capillaries that formerly surrounded it. The dermal papilla also moves upwards and away from its capillary tuft, so that in telogen the dermal papilla is still surrounded by a network of capillaries but not penetrated by any. The blood supply to the permanent (upper) zone of the follicle and the sebaceous gland remains essentially intact. At the next anagen phase, the new bulb grows down through the bundle of capillaries and develops inside it as a new vascular network regenerates (Montagna and Ellis, 1957; Sato et al., 1976). Thus the capillaries are independent of the upward and downward movement of the hair follicle during its growth cycles, and any changes that do occur to the blood vessels follow, rather than precede, changes occurring in the follicle (Allegra and De Panfilis, 1981). The blood supply to the dermal papilla, the hair follicle and the sebaceous gland form a single functional unit and would therefore seem to be important in the development and nutrition of the follicle. The most densely vascularized part of the follicle is at the level of the keratogenous zone in the upper limit of the bulb, and it is this level which is probably the most important region of exchange of metabolites such as glucose and oxygen necessary for growth (Montagna and Ellis, 1957). Ryder (1956) has shown that when sheep are injected with 35S-cystine the label appears first in the keratogenous zone and takes only about six minutes to do so. Clearly, this would be a route for the transfer of other materials, including those such as drugs (Baumgartner et al., 1989) and trace elements (Robertson, 1987) which are of forensic and clinical interest, to become incorporated within the hair. Another likely route of transfer would be through the basal germinative cells via the dermal papilla. There is some thought (Zviak and Dawber, 1986) that the follicle capillary network may be a channel for transferring glycogen from the outer root sheath, in which it is plentiful, to the dermal papilla for use in mitotic activity in the hair bulb. 1.3.10 Nerve Supply A network of nerves surrounds hair follicles from their base to their junction with the epidermis (Montagna and Parakkal, 1974). In particular there is a ring of nerve endings just beneath the branching point of the sebaceous duct (Montagna and Parakkal, 1974; Zviak and Dawber, 1986). Parallel nerve fibres form a palisade structure around the bulge region of large follicles and around the bulb of smaller vellus follicles. There is great variation in the patterns of the nerve endings, but those around the vellus follicles are always more precise than those of the larger follicles. In quiescent (telogen) follicles the nerve network collapses in the region formerly occupied by the degenerating bulb, but remains intact and active below the dermal papilla (Montagna and Parakkal, 1974). The nerve supply in alopecic skin is a sparse, thin and irregular net of nerve fibres without distinctive characteristics (Allegra and De Panfilis, 1981). The good supply of nerves to all follicles in the human skin regardless of their size indicates that the nerves have an important function, but since they are not needed for follicle growth it is likely that their role is a sensory one (Montagna and Parakkal, 1974). Montagna and Parakkal (1974) note that the follicles of the hairs in the human face and in the anogenital areas are particularly well supplied with nerves, and suggest that the shafts of hairs act as levers which amplify any movement and so provide a very sensitive response to the slightest touch. 14  Physiology and Growth of Human Hair 1.3.11 Muscles Each follicle has a bundle of smooth muscle, known as the arrector pili muscle (Pinkus, 1958) or erector muscle (Ryder, 1963). The lower end of the muscle is attached to the follicle just below the sebaceous gland duct, at the point which represents the lower extremity of the permanent zone of the follicle. The muscle runs at an angle upwards towards the skin, where it is attached to the papillary layer of the dermis. The follicle is at an angle to the skin surface and the arrector muscle is attached to the posterior side (i.e. the side under the slant—see Figure 1.2). Contraction of the muscle thus causes the follicle to stand more erect and produces the appearance on the skin surface known as ‘gooseflesh’ or ‘goose bumps’. This is a reaction to cold or fright which, while it may be important in many animals, does not appear to serve any useful purpose in humans. 1.4 The Hair The hair is the major component of the hair follicle commonly examined microsopically in forensic hair comparison. It is a long thin cylinder of keratinized cells and usually has three distinct cellular components: 1. a central medulla or core running along the central axis 2. the main component, the cortex 3. the cuticle (the outer covering). The general cellular arrangement in a mature hair is shown in Figure 1.4. Figure 1.4 Schematic cut-away section of a mature hair fibre showing the major cellular structures, cortex, medulla cells separated by air spaces, nuclear remnants in the medulla cells, macrofibrils—the aggregates containing the filaments and matrix (see Figures 1.7 and 1.8) 15 Forensic Examination of Hair 1.4.1 Medulla The medulla is formed as a column of cells that produce a protein that is distinct from the proteins of the cortex and cuticle in that it contains the amino acid citrulline (Rogers, 1959a, 1964; Harding and Rogers, 1971). During their formation, the cells collapse in such a manner that the medulla appears as a network of cellular connections with spaces and gaps that are filled with air (Auber, Figure 1.5 Human hair medulla, (a) A continuous medulla in a pubic hair. Note how the structure is visible where the mountant has replaced the air in the medulla near the worn tip (lower right), (b) Fragmented medulla, and (c) interrupted medulla, in which the cortex replaces the medulla when its formation in the bulb is interrupted, (d) Double medulla in beard hair 16  Physiology and Growth of Human Hair Figure 1.6 Scanning electron micrograph of a longitudinal hand-cut razor section of human beard hair showing the open structure of the medulla. Co, cortex; Cu, cuticle; M, medulla 1952; Rogers, 1964). The network in some mammalian species can be regular and diagnostic of species origin (Hausman, 1920; Wildman, 1954; Brunner and Coman, 1974). A medulla is not present in all human hairs and this is particularly so for very fine hairs, but when it is present the appearance can be described as irregular globular. In human hair the medulla can be continuous throughout the length of the hair (except for the tip and the root), or discontinuous (Hausman, 1920) (Figure 1.5). In coarser hairs it is generally continuous. When discontinuous, the medulla may be broken transversely at irregular intervals by cortical material (interrupted medulla) or it may only be present irregularly in very small amounts in the cortex (fragmented medulla) (Wildman, 1954). The human hair medulla is not large; it may only be one or two cells in diameter and usually not more than one-third the width of the hair shaft (Figure 1.5). The medulla diameter is generally small in the hair of the child, larger in middle age and largest in old age (Luell and Archer, 1964). Occasionally, hairs with a double medulla (Figure 1.5) are seen (Chowdhuri and Bhattacharyya, 1964; Montagna and Van Scott, 1958). The medulla may not necessarily be visible by light microscopy, particularly if the mountant used for microscopy has displaced the air from the intercellular and intracellular gaps. When the mountant does not infill, the medulla will appear dark when using transmitted light and very little structural detail will be seen (Stoves, 1957; Ryder, 1963) (see Figure 1.5) and the medulla can be mistaken for pigmentation (Griffith, 1848; Harding and Rogers, 1984). Random infilling of the medulla can produce what appears to be a discontinuous or a fragmented medulla, and this artefact should not be confused with real discontinuities which are generated as the result of periods of non-production of medulla cells in the follicle bulb. Infilling can occur where hairs are damaged (for example, cut) and it can also occur in intact hairs. Swift (1996) has shown that liquids can enter the hair fibre through adventitious holes (of the order of 0.5 nm in size) in the cell membrane complex, and also via exposed edges of the cuticle cells and a network of fine channels. The medulla that is filled with the mounting medium may be better visualized and its structure more clearly delineated using polarized light (Garn, 1951a). The morphology of the medullary tissue is dramatically different from the rest of the hair shaft components. There are many large intercellular and intracellular spaces and the protein material in the cells appears by light microscopy to be amorphous and unstructured (Matoltsy, 1953; Rogers, 1964). This appearance is the result of the apparent collapse and concentration at the cell periphery of the cell contents on transformation of the trichohyalin granules in the developing cells to the hardened, dehydrated medulla protein (Parakkal and Matoltsy, 1964; Rogers, 1964). Scanning electron microscope studies of human hair medulla clearly demonstrate the very open structure of the medulla (Figure 1.6). 17 Forensic Examination of Hair Clement et al. (1982) have found that the coalesced protein is present as macrofibrils, oriented randomly within the cells. The macrofilaments are composed of bundles of microfilaments. Medulla protein has an unusual composition in that it contains the amino acid citrulline in peptide linkage (Steinert et al., 1969). This amino acid is not normally found in proteins, but it is also present in inner root sheath protein (Rogers, 1964; Steinert et al., 1969). The medulla protein is remarkably insoluble, even more intractable than the keratins of the cortex (Matoltsy, 1953; Rogers, 1964), but the disulphide bonds found in keratin are virtually absent (Harding and Rogers, 1972b). In the case of medulla protein the insolubility is the result of extensive cross-linking of its peptide chains by isopeptide links between the side-chains of lysine residues and glutamic acid/glutamine residues (Harding and Rogers, 1971, 1976). This cross-link is the same type as that which forms to insolubilize the fibrin in a blood clot (Pisano et al., 1969). It also occurs in the inner root sheath protein and, in smaller amounts, in keratins of the cortex and cuticle (Rice et al., 1994; Zahn et al., 1994). In contrast again to the keratins, the medullary protein is very susceptible to digestion by proteolytic enzymes (Steinert et al., 1969; Harding and Rogers, 1971) and such enzymes will readily cause loss of medullary ‘structure’, a phenomenon seen in forensic casework with hair that has been subjected to decay conditions (Harding, H.W.J., unpublished observations). The role of the medulla is unclear. In larger (non-human) hairs where it occupies a large proportion of the hair shaft it may well because of its structure provide some mechanical stiffening, as well as increasing the thermal insulating properties of the hair. Clearly these are not essential functions in human hair (or in wool) which can grow without a medulla. It has been speculated that the medulla forms a channel for waste material. Kassenbeck (1981) has suggested that it provides a path for the evacuation of the water liberated by protein biosynthesis in the dividing cells of the follicle. It may well be that the function of the medulla is to maintain the diameter of the hair with the use of minimum resources and without producing excess weight (Auber, 1952; Ryder, 1973). Be that as it may, the medulla does appear to be the site (or at least a major site) of the ABO grouping ability of hair (Potsch- Schneider et al., 1986). 1.4.2 Cortex The cortex is composed of cells that are fusiform (spindle-shaped), about 80–100 µm long and 5–10 µm wide at their widest point. The cells are aligned parallel to the axis of the hair fibre and are closely packed in an interdigitating fashion. They are cemented together via intercellular contacts that are referred to as the intercellular membrane complex or cell membrane complex (CMC) (Fraser et al., 1972). The tensile strength of the hair depends in part on these contacts. In the fully formed hair the cortical cells contain some nuclear remnants (Fraser et al., 1972) and also some pigment granules, but they are mostly filled with keratin macrofibrils of about 0.1–0.2 µm in diameter. The macrofibrils are oriented longitudinally in the cortical cells and are thus also parallel to the axis of the hair. Each macrofibril is composed of keratin microfibrils (now called keratin intermediate filaments, or keratin IF) embedded in a matrix of sulphur-rich proteins (now called keratin-associated proteins, or KAP). The keratin IF proteins and matrix (KAP) proteins in the cortex are not single protein types but are each mixtures of keratin proteins. The proteins are characterized by a high level of sulphur compared to most proteins. In their natural state in the hair they are insoluble in water because they are highly cross-linked by disulphide bonds between adjacent cysteine residues in the protein chains. This insolubility presents a major difficulty in studying the keratin proteins. Solubilization (and hence extraction from the hair) requires the breaking of the disulphide bonds. This is commonly achieved by reduction of the bonds to cysteine residues using mercaptoethanol in 8M urea, followed by alkylation with iodoacetate of the cysteine residues to S-carboxymethylcysteine to prevent disulphide bond re- formation. Although much of what is known about hair keratins comes from studies on wool (Powell and Rogers, 1986), considerable work is now being done to characterize the proteins of human hair (see, for example, Baden, 1981; Gillespie, 1991; Yu et al., 1993) 18  Physiology and Growth of Human Hair Figure 1.7 A diagrammatic representation of the three dimensional arrangement of keratin IFs and the matrix of KAPs. The overall structure is sometimes referred to as the filament-matrix complex or the keratin complex. Modified from ‘The role of keratin proteins and their genes in the growth, structure and properties of hair’ by B.C.Powell and G.E.Rogers, in Formation and Structure of Human Hair, edited by P.Jolles, H.Zahn and H.Hocker, © 1997 Birkhauser Verlag Basel/Switzerland and it is clear that there are similarities and homologies (Marshall, 1983; Gillespie, 1991; Yu et al., 1993). The sulphur-rich matrix (KAP) proteins are amorphous and comprise about 40 per cent of the protein content of the cell. They have been referred to as ‘high-sulphur’ (HS) proteins and are characterized by a half-cystine content of about 27 mole per cent (Gillespie and Marshall, 1981). The seven protein fractions identified have a molecular weight range by sodium dodecyl sulphate (SDS) gel electrophoresis of 26,500–43,000 (Marshall, 1983). There are two classes or types (Type I and Type II) of the IF proteins (also known as ‘low-sulphur’ or LS proteins). Eight components have so far been identified (Gillespie, 1991) and as a group they have fewer half-cystine residues (about 8 mole per cent) than the KAP (Gillespie and Marshall, 1981) and a molecular weight range by SDS gel electrophoresis of 55,500–76,000 (Marshall, 1983). They are fibrous proteins with about 50 per cent a-helical content (Baden, 1981). The Type I proteins are acidic whereas the Type II are neutral-basic (Steinert and Freedberg, 1991). The amino acid sequence of one human hair Type I IF protein has been published (Yu et al., 1993). A third group of hair keratin proteins, the glycine- and tyrosine-rich proteins (‘high glycine/ tyrosine’, or HT proteins), which is found in the matrix of wool (and mouse hair), has not been found in matrix proteins extracted from human hair (Gillespie and Marshall, 1989). The electophoretic patterns of extracted proteins from human scalp hair are the same as those for hairs from other body regions of an individual (Gerhard and Hermes, 1987). Polymorphic variations of the proteins are seen between individuals and inheritance of the variants has been demonstrated (Baden, 1976; Yu et al., 1993). The fundamental unit of the keratin IF is formed from the pairing of the two IF protein types, I and II. Coiled-coils of these proteins are grouped together in the 8–10 nm units of the intermediate filament (microfibril). It has been estimated that keratin IFs contain 32 keratin protein chains within the cross- sectional area of the filament (Steinert, 1993). The helical proteins in the IFs are surrounded by the KAP proteins as a matrix or ‘cement’ (Figure 1.7), to which they are coupled by hydrogen and ionic bonding as well as covalent disulphide cross-links. It is not known what proportion of the disulphide bonds are intrachain and interchain. It is the cross-linking of each of these IF and KAP families together in the cell that produces the tensile strength and general toughness of hair. (It takes about three times as much force to break a hair as it does to pull it out.) Nevertheless, when hair is wet it is possible to stretch it, especially if it is heated. This comes about by the disruption of some of the chemical bonds (particularly ionic and hydrogen bonds) and the sliding of the polypeptide chains with respect to each other. The deformation will gradually disappear as the structures more or less return to their original positions. More drastic changes to the form of the hair can be achieved if the covalent disulphide bonds are broken (for 19 Forensic Examination of Hair example, under reducing conditions), the wet hair stretched and the bonds allowed to re-form in new positions. This is the basis of artificial waving (perming) in which the new bonds will more permanently hold the hair in its modified situation. It is now known that the keratin IF belong to the superfamily of proteins that form 8–10 nm diameter filaments in the cytoplasm of many epithelial cells, where they play the structural role of ‘cytoskeleton’ (Fuchs and Weber, 1994). The keratin IF are an elaborate extension of this role (Steinert and Freedberg, 1991). The manner in which the IFs are packed in the macrofibrils gives rise to two main types of cortical cells in hairs. These cell types are termed the orthocortex and the paracortex (Mercer, 1961; Fraser et al., 1972) and their distribution in the fibre can determine its form; for example, the crimp in wool fibres (Fraser and Rogers, 1955). The orthocortex is always on the outer side of the curve of the crimp. Intermediate types, particularly the mesocortex (or heterotype), are known (Fraser et al., 1972). The different cell types were originally detected by their differential reaction to staining with dyes (Horio and Kondo, 1953). It is known that this is the result of the protein composition and IF packing arrangements, but the reason for it is not understood. In paracortical cells the IFs are arranged mainly in quasi-hexagonal close-packing within the macrofibrils, with the matrix prominent in the interfilamentous space. In orthocortical cells the IFs are closer together and inclined to the cell axis, entwined in a rope-like fashion to produce a cylindrical lattice (Rogers, 1959b; Fraser et al., 1972). The IF packing in orthocortical cells produces a characteristic fingerprint-like ‘whorl’ in electron microscopy cross-sections (Fraser et al., 1972). In the mesocortical cells the IFs are packed in even more regular and extensive hexagonal arrays than are observed in the paracortex (Bones and Sikorski, 1967). The molecular basis for these packing modes is unknown, but it is likely that they are the result of quantitative or qualitative differences in the distribution of hair keratin proteins. All three types of IF packing can be recognized in electron micrographs of transverse sections of human hair, although the differences between the types are not as marked as seen in wool, and the chemical differences can not be so easily defined (Swift, A.J., personal communication). Fraser et al. (1972) have reported the ‘whorl’ orthocortical pattern in human hair (Figure 1.8), but noted that this was combined with a high proportion of high-sulphur (KAP) proteins (which is more a characteristic of paracortical material). Kassenbeck (1981) has made similar observations and has also reported hairs with mainly mesocortical structure but with orthocortical cells at the periphery adjacent to the cuticle. According to Swift (1977 and personal communication), straight Mongolian hair (which is basically circular in cross-section) is all paracortex, curly Caucasian hair has a core of paracortex with about 5 per cent orthocortex on the perimeter forming a layer about one cell thick and always on the outer side of the curl, and very curly Negroid hair (which is elliptical in cross-section) has a bilateral distribution with the orthocortex also on the outside of the curl, as occurs in wool. In some hairs small structures called cortical fusi occur in the cortex (Hausman, 1932). The fusi appear dark by transmitted light microscopy and may be mistaken for pigment granules (Harding and Rogers, 1984). That they are not pigment but are small air inclusions is demonstrated by their bright appearance by reflected light (Schwinger and Pott, 1977). A close study of their shape (they are fusiform, i.e. spindle-shaped, and therefore have pointed ends) should also help to distinguish them from the more rounded pigment granules (Noback, 1951). Fusi originally form as small fluid- filled spaces between the developing cortical cells when the cells are soft and pliable in the bulb region. As the cells move up the follicle they harden and keratinize, the hair dries out and the fluid is replaced by air (Hausman, 1932; Noback, 1951). There does not seem to be any genetic basis for the occurrence of fusi. They may occur throughout the length of the hair shaft but they are seldom seen at the tip. Frequently they are found just above the root (Noback, 1951). A possible explanation for this is that the fusi may be formed by mechanical forces as the result of flexing of the shaft at this point (which would correspond approximately to skin level when the hair is still attached to the follicle). In coloured hairs the cortical cells contain pigment granules in addition to the keratin fibrils. These granules contain the pigment melanin and so are sometimes also called melanin granules. They are ellipsoidal in shape, about 1 µm in length and 0.3–0.4 µm in diameter (Birbeck et al., 20  Physiology and Growth of Human Hair Figure 1.8 Electron micrograph of a stained transverse section of a keratinized human hair’s cortical cell, a region called the orthocortex. The packing mode of the keratin IF (microfibrils) and matrix into macrofibrils has the appearance of ‘whorls’ that resemble fingerprints. Bar = 0.1 µm. (Courtesy of Dr. L.N.Jones) 1956; Swift, 1977), but the size and shape are quite variable (Zviak and Dawber, 1986). Generally they are situated in the inter-macrofibrillar matrix with their long axis parallel to the length of the hair (Swift, 1977). There are two types (and colours) of pigment involved; the black-brown eumelanins, giving dark colours, and the reddish yellow phaeomelanins, giving light colours (Ortonne and Thivolet, 1981). Hair pigmentation is discussed in more detail in section 1.10. The colour of hair is due not only to the colour, density and distribution of the pigment granules in the cortex (Hausman, 1927), but also to the actual amount of melanin polymer within each granule (Swift, 1977). In human hair there tend to be more granules towards the periphery of the cortex than towards the centre (Swift, 1977) (Figure 1.9). They are sometimes also found in the medulla, but not usually in the cuticle of scalp hair (Hausman, 1927; Swift, 1977; Robbins, 1988). There are fewer coloured granules in greying hair and none in white hair (Szabo, 1965). Nuclear remnants can still be seen in fully keratinized cortical cells (Birbeck and Mercer, 1957a; Roth and Clark, 1964; Swift, 1977; Seta et al., 1988), but the DNA of the original chromatin is not visible (Rogers, 1969) and there has been some conjecture as to whether DNA is completely removed or broken down (Swift, 1977). The early work of Downes et al. (1966) suggested that the DNA was broken down during keratinization and resorbed, but Kalbe et al. (1988) and Schreiber et al. (1988) have shown that high molecular weight (genomic) DNA (>20 kb) is present in human hair and can be extracted from it. The studies by Kalbe et al. (1988) on wool cells show that although some of this DNA would come from the cuticle, it is likely that some is also from the cortex since this comprises the major part of the hair shaft. The amount extractable from a single 21 Forensic Examination of Hair Figure 1.9 Transverse section of a human pubic hair showing the higher concentration of pigment granules towards the periphery. Note that no tissue is present in the medulla shaft, though, is very small; von Beroldingen et al. (1989) found a level of 0.2–4 pg/cm, an amount which is too small for conventional DNA typing but which can be analyzed for HLA-DQa using polymerase chain reaction amplification (Higuchi et al., 1988). Mitochondrial DNA remains sufficiently intact in hair shaft cells to allow analysis by sequencing after amplification (Vigilant et al., 1989; Hopgood et al., 1992; Wilson et al., 1995). 1.4.3 Cuticle The outer layer of the hair shaft is called the cuticle. It is composed of flattened, imbricated scale cells (i.e. cells that overlap like tiles on a roof). The cells overlap both longitudinally and laterally to surround the hair completely and hold the cortex together. They slope outwards, their edges pointing towards the tip of the hair. In the follicle these edges interlock with opposing scales of the cuticle of the inner root sheath, helping at least in part to hold the hair in place in the follicle (Montagna and Van Scott, 1958; Straile, 1965). Thus pulling a growing hair with a quick, sharp tug will sometimes yield a hair root with inner root sheath material still attached (Ludwig, 1967). The cuticle of a human hair forms from a single layer of cells in the follicle. In the mature hair the cuticle cells are roughly rectangular in shape (Kassenbeck, 1981), about 50–60 µm long and about 0.5 µm thick (Swift, 1981). However, they overlap to such an extent that only about one-sixth of each cell is visible on the surface (Ryder, 1963), and the cuticle is effectively multilayered with a thickness of about six cells (about 3–5 µm) and scale edges about 5 µm apart (Swift, 1981). Cuticle cells form a pattern which can be visualized microscopically on the hair surface and is called the hair scale pattern. The pattern differs between species and can be used for species identification (see Noback, 1951; Wildman, 1954; Brunner and Coman, 1974). Using Wildman’s nomenclature the pattern for human hair is described as ‘close wave’, but the pattern can change with changes to the speed of growth of the hair (Kassenbeck, 1981). In spite of these changes, the scale counts (which are a measure of how far apart the edges are) are relatively constant for scalp hair for an individual providing sufficient counts are made, but they can vary between individuals (Gamble and Kirk, 1940). The scale count also varies on an individual depending on site, the count being significantly smaller (i.e. the scales further apart) for scalp hair than for hairs from nine other body sites, such as pubic, facial and chest. Smaller scale numbers are seen for the scalp hairs of younger people than older, and for facial, axillary and abdominal hairs of females as compared to hairs from the same sites from males (Wyatt and Riggott, 1977). The scale pattern of human hair corresponds to the edges of the cuticle cells (Kassenbeck, 1981). When the hair emerges from the skin, these edges are relatively smooth, and the cell surfaces are also relatively smooth, although closer inspection may reveal some marks imprinted on them by pressure from the IRS as the cells were keratinizing and hardening in the follicle (Kassenbeck, 1981; Swift, 1981). 22  Physiology and Growth of Human Hair Figure 1.10 Light micrographs showing (a) the typical human scale pattern on a (white) scalp hair, and (b) a worn tip end of scalp hair from which the cuticle has been lost and the cortical cells are starting to separate and fray Figure 1.11 Scanning electron micrographs of human scalp hair showing the difference in the detail of the scale and pattern at different points along the hair shaft. Three different hairs are used for illustration; in all cases the tip end is towards the right, (a) Near the root. (b) Mid-shaft. (c) Near the tip The cuticular surface of the fully developed hair is extremely hard when dry and protects the softer cortex from wear and tear (Swift, 1981). Nevertheless, the cuticle itself is damaged by processes such as weathering, combing, brushing, washing, and abrasion against other hairs, and the scale edges chip away (Swift, 1981). This causes the scale edges to become more irregular and produces the pattern typically seen for human hair (Figure 1.10). This phenomenon becomes more extensive the more the hair is exposed to insult. Since the hair shaft is dead tissue and cannot repair itself, greater damage will be seen towards the tips of hairs as compared with near the root (Bottoms et al., 1972; Swift and Brown, 1972; Montagna and Parakkal, 1974; Swift, 1977). This does not change the overall pattern (Figure 1.11), but it does affect the detail of it (Swift, 1981) and has ramifications with regard to the comparison of patterns (Swift, 1977). If the damage is extensive 23 Forensic Examination of Hair Figure 1.12 Electron micrograph of a stained transverse-section of the cuticle region of a human hair fibre. Note the three overlapping cuticle cells (1–3) external to the cortex (Co). The cell membrane complex ‘CMC’ (arrows) is seen between the cells and is thinner at the junction of the cortex and the cuticle. Each cell is filled with hardened proteins but three layers can be distinguished in the cells, the exocuticle (ex) with the outermost ‘A’ layer and the endocuticle (en). Bar = 0.1 µm. (Courtesy of Dr. L.N.Jones) and ongoing it will reduce the thickness of the overlapping cell layer eventually to the point where there is no longer a cuticle, and the cortex will be exposed (Swift and Brown, 1972). Without the protection of the cuticle the hair feels rough and the cortex frays and falls apart (Figure 1.10), with the formation of split tips and brush ends (Price, 1981). Three layers are distinguishable by ultrastructural analysis of cuticle cells. They are the endocuticle (on the inner side), the exocuticle (on the outer side), and a narrow layer, called the ‘A’ layer, on the outer edge of the exocuticle. These layers are clearly delineated in electron micrographs by the density of their electron staining (Figure 1.12), with the endocuticle being the least dense and the ‘A’ layer the most (Rogers, 1959c; Fraser et al., 1972; Swift, 1977). The cuticle cell contents are amorphous; filaments are not visibly prominent but lamella structures resulting from fused granules can be seen (Swift, 1981). The proteins of the exocuticle are cysteine and glycine-rich proteins (Fraser et al., 1972) containing on average 30 mole per cent half-cysteine. There are at least two families of proteins (MacKinnon et al., 1990; Jenkins and Powell, 1994) and the gene for one of these (KAP5) is known for human hair (MacKinnon et al., 1991). The endocuticle also consists mainly of protein, but this contains very little cysteine and large amounts of acidic and basic amino acids (Swift, 1981). Pigment granules are not normally found in the cuticle cells of human scalp hair. Nuclei, or the remnants of them, can be seen in the endocuticle (Swift, 1977; Kassenbeck, 1981). The nuclear DNA is not visible but it is apparently not completely degraded. Kalbe et al. (1988) have 24  Physiology and Growth of Human Hair shown that high molecular weight DNA (>20 kb) can be extracted from isolated cuticle cells of merino wool and thus presumably at least some of the similar-sized DNA they also extracted from human hair shafts would have been of cuticular origin. The ‘A’ layer (which is about 110 nm thick) contains extremely high levels of sulphur (1 in every 2.7 amino acids is present as a half-cystine), and this results in the unusual hardness of the hair surface (Swift, 1981). Each cuticle cell is completely surrounded by an epicuticle (Bradbury, 1973). This is a hydrophobic membranous layer about 10 nm thick. It forms the immediate outer surface of the hair. It is resistant to chemical and enzymic attack, presumably because its protein is highly cross-linked by both disulphide bonds and isopeptide bonds (Zahn et al., 1994). The hydrophobicity is due to a monomolecular layer of a C21 saturated fatty acid, 18-methyl-eicosanoic acid (18-MEA) (Evans et al., 1985) covalently associated with the protein (Zahn et al., 1994). The 18-MEA is also part of the intercellular cement layer (d-band) which binds the overlapping cuticle cells. Damage to the cells causes splitting of this cementing layer and exposes a fresh cuticle cell surface with its 18-MEA layer intact. The cuticle has functions other than just protecting the cortex. The hair surface shows ‘directional friction’ (friction is less going in the direction from root to tip than in the opposite direction) due to the imbricated arrangement of the scales (Swift, 1977). This phenomenon helps the hairs remove dentritis and irritants from the skin, and it also assists in keeping the hairs aligned (and thus not tangled and matted). The layer of 18-MEA acts as a boundary lubricant in this regard, contributing to the coefficient of friction (Swift, A.J., personal communication). The cuticle plays a large part in our perception of the ‘feel’ or ‘lift’ of the hair because of its effect on how the hair bends. The multilayering of the cuticle can cause it to be a large proportion of the overall diameter of the hairs, especially fine ones, and it therefore can make a large contribution to the hair stiffness. This effect is particularly seen in hairs of non-circular cross-section (for example, Negroid hairs) where the preferred bending will be in the direction of the minor axial diameter (Swift, A.J., personal communication). 1.5 Molecular Biology of Hair Growth 1.5.1 Structural Proteins of the Hair Follicle and Their Genes The Complexity of Keratin Proteins and the Nomenclature Problem The understanding of the complexity of hair keratin proteins as large gene families has increased enormously in recent years through the advent of gene cloning. Most of this advance in knowledge has been in studies of the genes for wool keratins, but the findings are largely applicable to human hair. Human hair genes have been studied directly as well (Yu et al., 1993). The keratin intermediate filament (keratin IF) proteins consist of two families, Type I and Type II (Figure 1.13); each of these is known to contain four related proteins which are referred to as the low- sulphur protein group in the older literature. As explained earlier (section 1.4.2), there is an obligatory pairing of one Type I and one Type II chain in the structure of the keratin IFs. Eight Type I hair IF sequences are now known from a number of species; five in their entirety and three in large part. These include one complete human sequence (Yu et al., 1993), two complete sheep sequences (Dowling et al., 1986; Wilson et al., 1988) and, for a mouse, three complete (Bertolino et al., 1988; Winter et al., 1994) and two partial (Tobiasch et al., 1992a; Winter et al., 1994) sequences. There are four Type II keratins in hair and there is another minor component in human and bovine hair but apparently not in sheep wool. Four sheep sequences (Sparrow et al., 1989, 1992; Powell et al., 1992; Powell and Beltrame, 1994) and two mouse sequences (Yu et al., 1991; Tobiasch et al., 1992a, 1992b) are known. The keratin proteins of the hair matrix (keratin-associated proteins, KAPs) are a more complex group and again they have been classified according to amino acid composition (Table 1.1). Part of 25 Forensic Examination of Hair Figure 1.13 The primary structure (amino acid sequences) of Type I and Type II hair intermediate filament keratin proteins. The sequences are sheep because complete sequences of human are not yet available. Nevertheless, the sequences are known to be homologous with human. The consequent secondary structure of an a-helical region, shown in bold, is also highly conserved between species including human. The sequences outside of the helical domains are the non- helical N- and C-terminal regions. The conventional one-letter designations are used for the amino acids. Data from Sparrow et al. (1989) and Dowling et al. (1986) this complex group is a very large superfamily with half-cystine contents that vary from 12 to 40 mole per cent. This superfamily is arbitrarily divided into two groups; those with half-cystine contents of up to 30 mole per cent (called high-sulphur proteins, or HS proteins) and those with greater than 30 mole per cent half-cystine content (referred to as ultra-high sulphur proteins, or UHS proteins). Each of these groups consists of several families. In some of the HS and UHS families there are tandem copies of the pentapeptide motif, cys-cys-arg/gln-pro-ser/thr. A family of UHS proteins has been found in the exocuticle region of the hair cuticle (MacKinnon et al., 1990). The other fraction of the matrix is a class of proteins prevalent in merino wool which have high contents of glycine and tyrosine (the high glycine/tyrosine, or HT proteins). These proteins are very low in abundance or absent from human hair (Gillespie, 1991). Two proteins of this class, referred to as Type I, have similar amino acid compositions but are not homologous to each other or with the Type II family, which is a large family of homologous proteins possibly made up of 20–30 members. Nomenclature for the keratin proteins and their genes is confusing, with new and different names accorded to proteins or genes as research proceeds. The old naming system of low-sulphur, high- sulphur and high glycine/tyrosine proteins originated from wool keratin proteins (Fraser et al., 1972), but it has become restrictive and cumbersome. This became particularly apparent when the epidermal keratin IFs were sequenced and were catalogued by Moll et al. (1982). No provision was made to incorporate the wool low-sulphur keratins when it became clear that they are related to the epidermal keratin IF (Hanukoglu and Fuchs, 1982; Weber and Geisler, 1982). The old nomenclature has continued to be applied to the wool keratin IF proteins, and totally different names have been arbitrarily given to the hair IF genes characterized from various species. The system for epidermal keratin IF (Moll et al., 1982) classifies the Type II keratins as K1-K8 and the Type I keratins as 26  Physiology and Growth of Human Hair Table 1.1 Amino acid content (residues %) of wool compared with separate protein components K9-K19 (in the genetic databases they are listed under the symbol KRT). New Type I IF keratins can be sequentially added as K20 onwards, but no simple extension is available for the Type II IF keratins. To retain the present Kn format, the Type II numbering would have to jump from K8 to beyond K21, and those in between would be Type I keratin IF, a cumbersome arrangement. This confusion has led to a proposal (Rogers and Powell, 1993; Powell and Rogers, 1994) for a revised nomenclature still linked to the scheme of Moll et al. (1982) and which incorporates all keratin IF. The families of hair keratin-associated proteins can be classified with a related system. In the proposed system (Table 1.2), the term Km.nxpL identifies keratin IF (K being replaced with KRT for gene symbols in the databases). The families of high-sulphur, ultra-high sulphur and high glycine/tyrosine hair proteins are distinguished by the term, KAPm.nxpL, indicating keratin- Table 1.2 A unified nomenclature for keratin proteins 27 Forensic Examination of Hair associated proteins (KAP being replaced with KRTAP for gene symbols in the databases). For the keratin IF the ‘m’ symbol is either ‘1’ or ‘2’, denoting Type I or Type II IF. For the keratin-associated proteins ‘m’ is any number denoting a family or a unique protein. The other symbols denote component number (n), variant (x), pseudogene (p) and ‘like’ (L). New genes are awarded the next available ‘n’ value and are readily integrated into the system. Where genes are different they can be given unique assignments but, for others, particularly from different species where more data might be needed before names can be confidently assigned, temporary names would be distinguished by an ‘L’ suffix. These systems can incorporate an unlimited number of keratin IF and KAP sequences and have already been implemented for sheep wool keratin proteins (Fabb et al., 1993; Fratini et al., 1993, 1994; Parsons et al., 1994b; Powell and Beltrame, 1994; Powell et al., 1995). In this system K14, a Type I IF keratin, would be K1.14; K6b, a Type II keratin, would be K2.6b. The cataloguing of the keratin IFs and KAPs requires the merging of data from different species into a logical nomenclature. The major criteria for common identity which can reasonably be applied are sequence similarity and pattern of expression. To illustrate the principal features of the suggested new nomenclature, some examples of assignments for the keratin IF and hair KAP genes and proteins are given in Tables 1.3 and 1.4. The Structure and Organization of Hair Keratin Genes The number of keratin genes involved in hair growth is not precisely known but is probably between 50 and 100. One of their major features is that they are clustered in families with individual genes usually only 5–10 kilobases (kb) apart in the genome. Another is that the IF genes are typical vertebrate genes in which the coding part is fragmented into short segments of coding sequence (exons), separated by often longer segments of non-coding intervening sequences (introns). In contrast, all the members of the matrix-type proteins so far sequenced have no introns. This is an Table 1.3 Keratin intermediate filament proteins 28  Physiology and Growth of Human Hair Table 1.4 Intermediate filament associated proteins unusual feature for vertebrate genes, of which there are only a few examples, such as the histone genes, which lack introns. By comparison, the genes of hair matrix proteins are very large families. In the following discussion, genes are given in italics whereas proteins are given in plain text. Genes for Keratin Intermediate Filament (IF) Proteins The structure of the hair keratin IF genes is very similar to that of the epidermal genes. The Type I genes contain six introns and are 4–5 kb in size (Figure 1.14), whereas the Type II genes contain eight introns and are larger, 7–9 kb in size (Wilson et al., 1988; Kaytes et al., 1991; Powell et al., 1992; Powell and Beltrame, 1994). 29 Forensic Examination of Hair Figure 1.14 (a) An outline of the exon-intron structure of the coding region of a Type I hair keratin gene. The cross-hatched regions represent the exons and the intervening solid lines indicate the positions of the introns. The respective positions of the initiating and terminating codons, ATG and TAG, are shown, (b) Diagram of the IF protein molecule outlining the a-helical domain consisting of four sub-domains, 1A, 1B, 2A, 2B, with N- and C-terminals that are non-helical. The approximate positions of introns in the DNA sequence are indicated by the vertical arrows The Type I and Type II epidermal genes map to different chromosomes (Lessin et al., 1988; Romano et al., 1988; Rosenberg et al., 1988; Nadeau et al., 1989; Popescu et al., 1989; Fries et al., 1991; Hediger et al., 1991) except for the Type I gene, K1.18, which maps to the Type II locus (Waseem et al., 1990; Yoon et al., 1994). Linkage between the hair and epidermal keratin genes has been established in mice (Compton et al., 1991) and other data are beginning to reveal the long-range organization of the keratin Type II locus (Powell and Beltrame, 1994; Yoon et al., 1994) in more detail. From these data the locus containing most, if not all, of the Type II IF hair and epidermal genes is possibly 500–600 kb in size, containing 20–30 genes. A 100 kb segment of DNA from the sheep keratin Type II locus has been characterized (Powell and Beltrame, 1994) in which six Type II genes, three hair and three hair-related, have been mapped. Since genes encoding three of the four sheep wool Type II proteins are clustered within 40 kb and flanked by hair-related genes, it is possible that hair genes are located in a domain that contains all the regulatory controls required for hair gene expression. At least one of the hair-related genes appears from its intact structure to be capable of encoding a functional protein, although no expression was detected in the hair follicle (Powell et al., 1993). The eight known epidermal-type genes, K2.1-K2.8, map to human 12q13 (Yoon et al., 1994) and mapping data have revealed the probable existence of several related genes, possibly hair genes. The hair and epidermal Type I keratin genes also are linked like the Type II genes (Compton et al., 1991), but information is limited on the size of this group of linked genes. Genomic clones showing linkage of two to three hair and hair-related genes have been described (Powell et al., 1986; Kaytes et al., 1991) and epidermal keratin genes, K1.14 and K1.16, are linked within 10 kb of each other (Rosenberg et al., 1988, Savtchenko et al., 1990) and the K1.13, K1.15 and K1.19 genes are located within a 55 kb segment of DNA (Filon et al., 1994). Genes for Keratin (Intermediate Filament)-Associated Proteins (KAPs) In the past few years some interesting features of the KAP genes encoding the proteins of the matrix have emerged. There are at least ten families of KAP genes and each gene is rather small, between 0.6 and 1.5 kb in size. The absence of introns is a rare occurrence in vertebrate genes and the hair KAP genes would constitute one of the largest groups of intron-less genes known. 30  Physiology and Growth of Human Hair The human KAP5 gene family that is expressed in the hair cuticle may contain as many as ten genes (Powell et al., 1991) located at two sites on human chromosome 11, at 11p15 and 11q13 (MacKinnon et al., 1991). Of the other KAP gene families that encode cysteine-rich hair proteins (KAP3, 4, 9, and 11), sufficient data have been reported only for the KAP9 family, where three genes are located in an 18 kb piece of DNA (McNab et al., 1989). The KAP6 gene family that encodes glycine/tyrosine-rich proteins comprises at least nine genes, and in the sheep genome they are located within a 1 Mb segment of DNA, three of them within 40 kb (Fratini et al., 1993). RFLP studies indicate that the KAP8 gene which encodes a different glycine/ tyrosine-rich protein is linked to this group (Parsons et al., 1994a) and located on sheep chromosome 1 (Wood et al., 1992). The location of the KAP7 gene is at present unknown. The findings of Parsons et al. (1994b) suggest that a KAP1 locus is linked to the locus for keratin Type I IF genes (KRT1) on sheep chromosome 11. They discovered the linkage of at least one KAP1 gene to growth hormone by RFLP studies and they deduced the linkage of the KAP1 and KRT1 loci from the linkage between growth hormone and the KRT1 locus. Linked KAP1 and KAP2 genes have been characterized