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AN72.1-AN73.3 | Integumentary System — Self-Directed Learning

Skin Histology: Layers and Appendages (AN72.1)

The skin (integument) consists of two main layers:

1. Epidermis — stratified squamous keratinised epithelium derived from ectoderm

Layers of the epidermis (deep to superficial — mnemonic: "Come, Let's Get Sun-Burned"):

Layer	Name	Key Features
C	Stratum basale (germinativum)	Single layer of cuboidal/columnar cells; contains stem cells + melanocytes; attached to basement membrane by hemidesmosomes
L	Stratum spinosum	8–10 layers of polyhedral cells linked by desmosomes; "prickle cells" (spines = desmosomal connections); site of Langerhans cells (immune surveillance)
G	Stratum granulosum	3–5 layers of flattened cells with basophilic keratohyalin granules + lamellar bodies (lipid-rich, create waterproof barrier)
S	Stratum lucidum	Present only in thick skin (palms, soles); a clear, pale-staining layer of dead, anucleate cells packed with eleidin
B	Stratum corneum	15–30 layers of flat, anucleate, keratin-filled dead cells (corneocytes); the actual physical and waterproof barrier

Thick skin (palms, soles): Has all 5 layers including stratum lucidum; no hair follicles; abundant eccrine sweat glands; fingerprint ridges (dermatoglyphics).
Thin skin (everywhere else): No stratum lucidum; hair follicles, sebaceous glands present.

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2. Dermis — connective tissue derived from mesoderm
- Papillary dermis: thin, loose connective tissue; papillae interdigitate with epidermal ridges; contains capillaries (nourish epidermis), Meissner's corpuscles (fine touch)
- Reticular dermis: thick, dense irregular connective tissue; collagen and elastic fibres (type I collagen dominant); contains Pacinian corpuscles (deep pressure/vibration), hair follicles, glands

Beneath the dermis: Hypodermis (subcutaneous tissue) — loose connective tissue + adipocytes; not part of the skin proper but part of the integumentary system.

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Skin Appendages:

Hair follicles:
- Each follicle has an outer root sheath (epidermal), inner root sheath, and the hair shaft (medulla + cortex + cuticle)
- Hair bulb at the base: matrix cells (germinative cells) + melanocytes (pigment the hair)
- Arrector pili muscle: smooth muscle; contraction → "goosebumps" (piloerection)
- Sebaceous gland: opens into the hair follicle (pilosebaceous unit); holocrine secretion (whole cell dissolves → sebum)

Sweat glands (two types):
- Eccrine (merocrine) sweat glands: simple coiled tubular glands; present over entire body surface; secrete watery, hypotonic sweat (thermoregulation); ducts open directly to skin surface
- Apocrine sweat glands: larger; open into hair follicles; present in axilla, groin, areola; produce thick, odourless secretion (becomes odorous after bacterial action); activated at puberty; apocrine secretion mechanism

Nails:
- Plate of hardened keratin; nail matrix (germinal matrix at base and sides) = site of nail growth; nail bed = epidermis under the nail plate; lunula = white crescent = visible part of matrix

Chromosome Structure and Classification (AN73.1)

Chromosome structure:

A human chromosome is a single, linear, double-stranded DNA molecule complexed with histone proteins:
- DNA (double helix) → wraps around nucleosome octamer (H2A, H2B, H3, H4 × 2) → nucleosome bead-on-a-string → 30 nm fibre → looped domains → chromatin fibre → chromosome (visible at metaphase)

Key chromosome anatomy:
- Centromere: primary constriction; where spindle fibres attach (kinetochore); divides chromosome into arms
- Short arm (p): from French petit
- Long arm (q): by convention
- Telomeres: repetitive sequences (TTAGGG) at chromosome ends; protect chromosome from degradation and fusion; shorten with each cell division (ageing clock)
- Secondary constrictions: additional constrictions in some chromosomes at nucleolar organiser regions (NOR) — chromosomes 13, 14, 15, 21, 22 → produce ribosomal RNA
- Satellite chromosomes: small knobs attached to secondary constriction by a thin stalk

Types of chromatin:
- Euchromatin: actively transcribed, loosely packed, pale-staining
- Heterochromatin: transcriptionally inactive, densely packed, darkly staining; constitutive (always condensed — centromeres, telomeres) vs facultative (can be activated — Barr body/inactive X)

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Chromosome Classification (Denver system / 1960 and Paris Convention 1971):
Based on arm ratio and centromere position:

Type	Centromere position	p:q ratio	Examples
Metacentric	At or near the middle	1:1 (equal arms)	1, 3, 16, 19, 20
Submetacentric	Slightly off-centre	Short p, longer q	2, 4–12, 17, 18, X
Acrocentric	Near one end	Very short p arm	13, 14, 15, 21, 22, Y
Telocentric	At the tip	No p arm	Not found in normal humans

Groups A–G (Denver classification):
- A (1–3): largest, metacentric/submetacentric
- B (4–5): large submetacentric
- C (6–12 + X): medium submetacentric
- D (13–15): medium acrocentric (NOR satellites)
- E (16–18): small; 16 metacentric, 17–18 submetacentric
- F (19–20): small metacentric
- G (21–22 + Y): smallest; 21–22 acrocentric (NOR); Y variable

Karyotyping: Technique and Applications (AN73.2)

Karyotyping = the process of preparing and analysing the complete chromosome complement of a cell, arranged in a standardised display (karyogram).

Technique:

Step 1 — Cell source:
- Peripheral blood lymphocytes (most common in postnatal diagnosis) — short-term culture (48–72 hours)
- Amniotic fluid cells (prenatal — amniocentesis at 15–18 weeks)
- Chorionic villi (prenatal — CVS at 10–12 weeks; faster result)
- Bone marrow (haematological malignancies)
- Skin fibroblasts (for some specific indications)

Step 2 — Cell culture:
Cells cultured in medium with phytohaemagglutinin (PHA) (stimulates T-lymphocyte mitosis) → cell division initiated

Step 3 — Arrest at metaphase:
Colchicine (or colcemid) added → binds tubulin → inhibits spindle formation → cells arrested in metaphase (chromosomes maximally condensed and visible)

Step 4 — Hypotonic treatment:
Cells placed in hypotonic solution (0.075 M KCl) → cells swell → chromosomes spread apart

Step 5 — Fixation:
Carnoy's fixative (methanol:acetic acid 3:1) → fixes chromosomes; lyses cell membrane

Step 6 — Spreading:
Cell suspension dropped onto slides → cells burst → chromosomes spread flat

Step 7 — Staining (banding):
- G-banding (Giemsa): most widely used; AT-rich regions stain dark (late replicating); GC-rich regions stain light; produces characteristic dark/light band pattern unique to each chromosome → allows identification
- Q-banding (Quinacrine): fluorescent; first banding technique; Y chromosome brightly fluorescent (Y body in interphase)
- R-banding: reverse of G-banding
- C-banding: centromeres and constitutive heterochromatin stain dark

Step 8 — Analysis:
Photomicrograph taken; chromosomes cut out (or digitally processed) and arranged in homologous pairs, numbered 1–22 + sex chromosomes → karyogram (karyotype image)

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Clinical Applications of Karyotyping:

Indication	Chromosome finding
Down syndrome	Trisomy 21 (47, +21)
Edwards syndrome	Trisomy 18 (47, +18)
Patau syndrome	Trisomy 13 (47, +13)
Turner syndrome	45,X (monosomy X)
Klinefelter syndrome	47,XXY
Cri-du-chat	46, 5p- (partial deletion of short arm of chromosome 5)
Chronic myeloid leukaemia	t(9;22) — Philadelphia chromosome — BCR-ABL fusion
Prenatal diagnosis	Amniocentesis/CVS at advanced maternal age (>35 years)

Indian epidemiology: Down syndrome birth prevalence in India is approximately 1/650–1/1,000 live births. Advanced maternal age is the dominant risk factor; prenatal diagnosis and genetic counselling are part of antenatal care protocols.

Lyon's Hypothesis of X-Inactivation (AN73.3)

Lyon's Hypothesis (Mary Lyon, 1961):

In females (XX), one of the two X chromosomes in each somatic cell is randomly inactivated early in embryonic development (around the blastocyst stage, ~16 cell stage). This inactivated X forms the Barr body (sex chromatin body).

Key principles:
1. Randomness: Inactivation is random — either the maternal X or the paternal X can be inactivated in any given cell
2. Permanence: Once inactivated in a cell, the same X remains inactive in all daughter cells (clonal propagation)
3. Dosage compensation: Ensures that X-linked gene expression is equalized between XX females and XY males (both effectively have one active X per cell)
4. Completeness (with exceptions): Most genes on the inactive X are silenced; however, some genes in the pseudoautosomal regions (PAR1, PAR2) and other regions escape inactivation — these are still expressed from both X chromosomes

Molecular mechanism of X-inactivation:
- XIST gene (X-inactive specific transcript) on the inactive X produces a non-coding RNA that coats the inactive X chromosome in cis
- XIST RNA recruits chromatin-modifying enzymes → histone H3 lysine 27 trimethylation (H3K27me3) and DNA methylation of CpG islands
- The result: the X becomes heterochromatic (Barr body) — compacted, transcriptionally silent

Barr body (sex chromatin):
- Visible as a dark-staining condensed mass at the nuclear periphery in female somatic cells
- Number of Barr bodies = (number of X chromosomes − 1)
- Normal female (XX) = 1 Barr body
- Klinefelter (47,XXY) = 1 Barr body
- Triple X female (47,XXX) = 2 Barr bodies
- Turner syndrome (45,X) = 0 Barr bodies

Clinical implications of Lyon's hypothesis:
1. X-linked recessive disorders in carrier females: Because inactivation is random, carrier females have a mosaic of cells expressing the mutant X and cells expressing the normal X. Most carriers are phenotypically normal because ~50% of cells express the normal allele. However, if inactivation is skewed toward the normal X, the carrier becomes symptomatic (manifesting carrier).
- Example: Carrier female for Duchenne muscular dystrophy may have mild muscle weakness if skewed inactivation leaves predominantly mutant X active.
2. Calico cats: The classic model of Lyon's hypothesis — orange/black coat colour gene is X-linked; inactivation in skin cells → patches of orange and black; females only (XX) can be calico.
3. Barr body count in forensic/clinical use: Buccal smear for Barr body count was historically used to determine sex in intersex individuals and athletes (now replaced by karyotyping/molecular diagnosis).

Reactivation: The inactive X is reactivated in germ cells before meiosis — both X chromosomes must be active for normal oogenesis.