---

1. Cytogenetic Disorders Involving Sex Chromosomes & Sex-Linked Disorders

Background: Lyonization (X-Inactivation)

1. Lyon hypothesis (lyonization): In females, only one X chromosome is genetically active. X inactivation occurs ~16 days post-conception. Either the paternal or maternal X is randomly inactivated in each cell, and the same X remains inactive in all daughter cells. This is mediated by the XIST long non-coding RNA, which "coats" the inactive X chromosome and silences its genes.
   • However, ~30% of genes on Xp and 3% on Xq escape X-inactivation - important for understanding Turner syndrome.
2. Y chromosome: Carries minimal genetic information beyond male sex determination, so extra Y chromosomes are well tolerated.

General Features of Sex Chromosome Disorders

• Often produce subtle changes, sometimes undetected at birth
• Infertility is a common feature, not diagnosable until adolescence
• Loss of sex chromosomal material is better tolerated than loss of autosomal material
• Extra X chromosomes cause progressively more severe phenotypes with increasing number

Klinefelter Syndrome (47,XXY) - see Topic 5 below

Turner Syndrome (45,X)

• Karyotype: ~57% have 45,X (complete monosomy); 14% have structural X abnormalities (isochromosome Xq, deletions); 29% are mosaics (45,X/46,XX)
• Pathogenesis: During normal development, ovaries develop normally in early embryogenesis but the absence of the second X leads to accelerated loss of oocytes. By age 2, the ovaries are reduced to fibrous "streak ovaries" - "menopause occurs before menarche"
• SHOX gene: The short stature homeobox (SHOX) gene at Xp22.33 escapes X-inactivation; normally two active copies exist in both sexes. Loss of one copy in Turner syndrome explains short stature
• Clinical features:
  • Short stature (most consistent somatic feature)
  • Primary amenorrhea and infertility (streak ovaries)
  • Webbing of the neck (pterygium colli)
  • Low posterior hairline
  • Cubitus valgus (wide carrying angle of arms)
  • Broad shield-shaped chest with widely spaced nipples
  • Congenital heart defects (coarctation of aorta, bicuspid aortic valve)
  • Lymphedema of hands and feet at birth
  • Renal anomalies (horseshoe kidney)
  • Intelligence is usually normal; some specific cognitive deficits (spatial reasoning)
• Mosaics (45,X/46,XX): May have minimal findings, present only with primary amenorrhea

Other Sex Chromosome Disorders

Sex-Linked Disorders

• Affect males almost exclusively (hemizygous - only one X)
• Females are carriers (heterozygous) and usually unaffected; may show mild features due to random X-inactivation
• Examples: Haemophilia A (Factor VIII deficiency), Haemophilia B (Factor IX deficiency), Duchenne muscular dystrophy, G6PD deficiency, colour blindness, Fragile X syndrome

• Both males and females affected, but males more severely
• Lethal in hemizygous males in some conditions (e.g., incontinentia pigmenti)
• Example: Fragile X syndrome (moderate penetrance in females)

• No male-to-male transmission
• All daughters of affected males are obligate carriers
• 50% of sons of carrier females are affected; 50% of daughters are carriers

---

2. Fragile X Syndrome

• Hypermethylation of the FMR1 promoter CpG island
• Histone deacetylation
• Transcriptional silencing of FMR1
• Absent FMRP protein (a ~80 kDa RNA-binding protein expressed in neurons and non-neural tissues)

• Moderate intellectual disability (cognitive impairment is the cardinal feature)
• Autism spectrum disorder (~2-3% of ASD patients have FXS)
• Long, narrow face with prominent forehead and jaw
• Large, protruding ears
• Macroorchidism (post-pubertal testicular enlargement)
• Hyperextensible joints, flat feet
• Behavioural features: hyperactivity, hand-flapping, temper tantrums, perseverative speech, poor eye contact

• FXTAS (Fragile X-associated tremor/ataxia syndrome) - late-onset in males
• FXPOI (Fragile X-associated premature ovarian insufficiency) - in females

---

3. Gaucher's Disease

Three Clinical Types

• Hepatosplenomegaly (splenomegaly often massive)
• Bone marrow infiltration causing anaemia, thrombocytopenia, leukopenia
• Bone pain and pathological fractures (Erlenmeyer flask deformity of distal femur)
• No neurologic involvement

1. Enzyme replacement therapy (ERT): Lifelong IV infusion of recombinant glucocerebrosidase (imiglucerase/velaglucerase)
2. Substrate reduction therapy: Oral inhibitor of glucosylceramide synthase (miglustat, eliglustat) - reduces glucocerebroside production
3. Emerging: gene therapy via hematopoietic stem cell transplant expressing normal glucocerebrosidase

---

4. Fluorescence In Situ Hybridisation (FISH)

1. Prepare metaphase spreads or interphase nuclei on a slide
2. Denature both probe and chromosomal DNA (strand separation)
3. Allow fluorescently-labelled probe to hybridise to its target sequence
4. Wash away unhybridised probe
5. Visualise under fluorescence microscope - positive hybridisation appears as fluorescent spots

• Locus-specific probes: Detect a specific gene or chromosomal region (e.g., HER2 amplification in breast cancer)
• Centromeric/alpha-satellite probes: Detect chromosome number using centromere sequences (e.g., chromosome enumeration in aneuploidy)
• Whole chromosome painting probes: Contain sequences from an entire chromosome; used for detecting translocations
• Telomere probes: Detect cryptic subtelomeric rearrangements

• Detection of chromosomal aneuploidy (e.g., trisomy 21, 18, 13; sex chromosome abnormalities)
• Detection of microdeletions (e.g., 22q11.2 deletion syndrome - DiGeorge/velocardiofacial syndrome - cannot be detected by conventional karyotype; requires FISH)
• Detection of translocations (e.g., BCR-ABL in CML)
• Gene amplification in cancer (e.g., HER2/neu, MYCN in neuroblastoma)
• Prenatal diagnosis on uncultured amniocytes (rapid)
• Preimplantation genetic diagnosis

• Can detect submicroscopic deletions and duplications (<5 Mb) invisible on G-banding
• Can be performed on interphase cells (no need for cell culture/metaphase)
• Higher resolution for specific loci

• Only identifies abnormalities at targeted loci (not a genome-wide screen)
• Cannot detect point mutations
• Requires prior knowledge of the suspected abnormality

---

5. Klinefelter Syndrome

Clinical Features

• Tall stature with increased soles-to-pubic bone length (elongated legs)
• Gynecomastia
• Reduced facial, body, and pubic hair
• Female fat distribution

• Small, firm testes (often only 2 cm) - most consistent feature
• Azoospermia/severe oligospermia → sterility (rare fertility in mosaics)
• Low serum testosterone; elevated FSH and LH
• Histology: hyalinisation of seminiferous tubules (ghost-like structures); prominent Leydig cells (apparent hyperplasia)

• IQ ranges from average to below average; verbal IQ deficit more common
• Increased risk of learning difficulties and language delay

• Type 2 diabetes and metabolic syndrome (insulin resistance)
• Mitral valve prolapse (~50% of adults)
• 20-30-fold increased risk of extragonadal germ cell tumours (especially mediastinal teratomas)
• Increased risk of breast cancer
• Increased risk of autoimmune diseases (SLE)

• Testosterone replacement from puberty onwards (improves virilisation, energy, bone density)
• Gynecomastia may require surgery
• Assisted reproduction (testicular sperm extraction + ICSI) possible in some patients

---

6. Glycogen Storage Diseases (GSD)

Major Types

Treatment

• GSD I: Frequent feedings + uncooked cornstarch (slow glucose release) to maintain normoglycaemia; avoid sucrose/fructose/galactose; multivitamins, calcium, Vitamin D. Empagliflozin (SGLT2 inhibitor) for GSD Ib (reduces neutrophil dysfunction). Liver transplantation for multiple or malignant adenomas.
• GSD II (Pompe): Enzyme replacement therapy - alglucosidase alfa (recombinant acid alpha-glucosidase); newer avalglucosidase alfa has higher mannose-6-phosphate tags for better uptake. Multidisciplinary team: physiotherapy, speech therapy, ventilatory support.
• GSD III: Uncooked cornstarch + high-protein diet (up to 3g/kg/day) to utilise intact gluconeogenesis; liver or cardiac transplantation in advanced disease.
• GSD V (McArdle): Aerobic exercise training; avoid intense exercise; sucrose supplementation before exercise.

---

7. Gene Mutations

Types of Gene Mutations

1. Point Mutations (Single Nucleotide Substitutions)

• Missense mutation: One nucleotide substituted by another, changing one amino acid to another. Example: β-globin point mutation (GAG→GTG) changing glutamate to valine → Haemoglobin S → sickle cell disease.
• Nonsense mutation: Point mutation creates a stop codon (UAA, UAG, UGA) → truncated/absent protein. Example: many mutations causing Duchenne muscular dystrophy.
• Silent mutation: Nucleotide change does not alter amino acid (due to degeneracy of genetic code) - no phenotypic effect.

2. Frameshift Mutations

• Insertion or deletion of one or two base pairs (not multiples of 3) → alters reading frame of all downstream codons → usually non-functional protein.
• Example: Most mutations in Duchenne muscular dystrophy are deletions causing frameshift.

3. Trinucleotide (Triplet) Repeat Mutations

• Amplification of a 3-nucleotide sequence beyond a threshold number.
• Dynamic mutations: Degree of amplification increases with transmission (especially in certain sexes) → anticipation.
• Examples:
  • Fragile X syndrome: CGG repeat in FMR1 (normal <55; full mutation >200)
  • Huntington disease: CAG repeat in HTT (normal <35; disease >40)
  • Myotonic dystrophy: CTG repeat in DMPK
  • Spinocerebellar ataxias: Various CAG repeats
• ~50 diseases caused by trinucleotide repeats; all involve neurodegeneration.

4. Splice Site Mutations

• Mutations at intron-exon boundaries disrupt pre-mRNA splicing → exon skipping, intron retention → abnormal protein.

5. Promoter/Regulatory Region Mutations

• Affect transcription factor binding → altered gene expression level without changing protein structure.

6. Large Deletions/Insertions

• Deletion of an entire exon or multiple exons.
• Example: Deletion mutations in dystrophin gene in Duchenne/Becker MD.

7. Copy Number Variants (CNVs)

• Amplifications or deletions of chromosomal segments containing multiple genes.
• Example: HER2 amplification in breast cancer; 22q11.2 microdeletion.

Functional Consequences

• Loss-of-function (LOF): Reduced or absent protein activity. Most autosomal recessive diseases result from LOF mutations in both alleles.
• Gain-of-function (GOF): Abnormal new function or overactivity. Example: dominant negative effect, constitutive receptor activation (e.g., RET mutations in MEN2).
• Dominant negative: Mutant protein interferes with the function of the normal protein from the other allele. Example: p53 mutations in Li-Fraumeni syndrome.

---

8. Gene Therapy

Approved/Clinical Applications (Table from Thompson & Thompson)

Vectors

• Retroviruses/Lentiviruses: Integrate into host genome → stable long-term expression. Risk of insertional mutagenesis (activating oncogenes). Used for haematopoietic stem cells.
• Adeno-associated virus (AAV): Does not integrate (forms episome); low immunogenicity; long-term expression in post-mitotic cells (neurons, hepatocytes, myocytes). Most widely used vector. Risk: limited cargo size (~4.7 kb).
• Adenoviruses: High efficiency transduction; episomal; strong immune response limits repeat dosing.

Strategies

1. Gene addition: Introduce functional copy of a defective gene (most common; for LOF diseases)
2. Gene correction: Edit the defective gene in its native chromosomal context (using CRISPR-Cas9)
3. Gene silencing: RNA interference (RNAi) or antisense oligonucleotides to suppress GOF mutations (e.g., Huntington disease)

Somatic vs. Germline Gene Therapy

• Somatic gene therapy: Targets specific cells (lung, liver, HSCs); changes are not passed to offspring. Currently approved and in clinical use.
• Germline gene therapy: Changes reproductive cells; would be inherited by future generations. No country currently permits this. Raises major ethical concerns regarding consent of future persons, enhancement vs. therapy, unpredictable long-term effects.

Induced Pluripotent Stem Cells (iPSCs) in Gene Therapy

1. Gene-corrected (by gene editing or gene therapy)
2. Induced to pluripotency → differentiated into the required cell type
3. Transplanted into the patient

---

9. Down's Syndrome (Trisomy 21)

Karyotypes

Pathogenesis

• <20 years: 1 in 1550 births

• 45 years: 1 in 25 births

Clinical Features

• Flat facial profile
• Oblique (upward-slanting) palpebral fissures
• Epicanthic folds
• Small, low-set ears
• Brushfield spots on iris
• Protruding tongue, small mouth (relative macroglossia)

• Leading cause of severe intellectual disability; IQ 25-50 in ~80%
• Hypotonia (neonatal)
• Alzheimer disease in virtually all patients >40 years (APP gene on chr 21)

• Endocardial cushion defects (most common)
• Atrial septal defects
• Ventricular septal defects
• Accounts for majority of early deaths

• 10-20-fold increased risk of acute leukaemia (both ALL and AML)

• Atlantoaxial instability (~10-20%)
• Hypothyroidism (increased incidence)
• Gastrointestinal: duodenal atresia, Hirschsprung disease
• Ophthalmologic: cataracts, refractive errors
• Recurrent infections (impaired T-cell function, thyroid autoimmunity)
• Cryptorchidism, hypospadias

Prenatal Screening

• First trimester: maternal serum β-hCG (elevated), PAPP-A (decreased), nuchal translucency ultrasound
• Cell-free fetal DNA in maternal blood (liquid biopsy): sensitive and specific, but confirmed by conventional cytogenetics (amniocentesis)

---

10. Lysosomal Storage Diseases (LSDs)

General Mechanism

Classification by Substrate Accumulated

Common Features

• Hepatosplenomegaly (phagocytic cells accumulate storage material)
• Progressive neurodegeneration (for disorders with CNS involvement)
• Coarse facial features (especially mucopolysaccharidoses)
• Skeletal abnormalities (dysostosis multiplex in MPS)
• Corneal clouding (MPS, mucolipidoses)
• Onset in infancy/childhood; progressive deterioration

Treatment Approaches

1. Enzyme replacement therapy (ERT): IV infusion of recombinant enzyme. Effective for Gaucher type 1, Pompe, Fabry, some MPS types. Cannot cross the blood-brain barrier → ineffective for CNS disease.
2. Substrate reduction therapy: Reduce substrate production (miglustat for Gaucher and Niemann-Pick type C).
3. Haematopoietic stem cell transplantation: Provides donor macrophages expressing normal enzyme. Effective if done early (before neurologic damage); used for Hurler syndrome.
4. Chaperone therapy: Small molecule chaperones stabilise misfolded enzymes (clinical trials for Gaucher, Fabry, some Tay-Sachs variants).
5. Gene therapy: Emerging; clinical trials ongoing.

---

11. Niemann-Pick Disease

Type A and Type B - Sphingomyelinase Deficiency

Type A (Severe, Infantile Neuronopathic)

• Complete sphingomyelinase deficiency
• Sphingomyelin accumulates in phagocytic cells and neurons
• Macrophages become stuffed with lipid droplets ("foam cells") → fine vacuolation/foaminess - "zebra bodies" on electron microscopy (concentric lamellar myelin figures)
• Organs most affected: Spleen, liver, bone marrow, lymph nodes, lungs (high phagocytic cell content)
• CNS: All levels affected (spinal cord, ganglia, brain); neurons enlarged and vacuolated
• Clinical: Progressive hepatosplenomegaly, failure to thrive, neurologic deterioration from infancy
• Cherry red spot in retina (similar to Tay-Sachs, as both involve ganglion cell swelling)
• Fatal: Death usually within first 3 years of life

Type B (Non-Neuronopathic)

• Mutant sphingomyelinase with some residual activity
• Organomegaly (hepatosplenomegaly) present
• No neurologic manifestations (unlike Type A)
• Patients survive into adulthood
• Diagnosis: Sphingomyelinase activity in leukocytes; molecular genetic testing

Type C - Cholesterol Transport Defect

---

12. Single Gene Disorders

Inheritance Patterns

Autosomal Dominant (AD)

• One abnormal allele sufficient to cause disease
• Heterozygote affected; vertical transmission through generations
• 50% risk to offspring
• Often due to gain-of-function (GOF) or dominant negative mutations; haploinsufficiency in some
• Examples: Marfan syndrome, Huntington disease, familial hypercholesterolaemia (heterozygous), neurofibromatosis, achondroplasia, BRCA1/2-related cancer syndromes

Autosomal Recessive (AR)

• Both alleles must be abnormal (homozygous or compound heterozygous)
• Parents are typically unaffected carriers
• 25% risk to offspring of two carriers
• Usually enzyme/structural protein deficiencies
• Examples: Cystic fibrosis, sickle cell disease, phenylketonuria, Tay-Sachs disease, Gaucher disease, all glycogen storage diseases (except some forms)

X-Linked Recessive

• Gene on X chromosome; affects hemizygous males
• Females (heterozygous) are usually carriers
• No male-to-male transmission
• Examples: Haemophilia A and B, Duchenne MD, G6PD deficiency, Fragile X (X-linked with modified dominance)

X-Linked Dominant (rare)

• Both males and females affected; males often more severely
• Examples: Fragile X syndrome (reduced penetrance in females), incontinentia pigmenti, Rett syndrome

Non-Mendelian Single Gene Disorders

1. Triplet Repeat Disorders

• Fragile X, Huntington disease, myotonic dystrophy, spinocerebellar ataxias
• Dynamic mutations, anticipation (see Topics 2 and 7)

2. Mitochondrial (Maternal) Inheritance

• Mutations in mitochondrial DNA (mtDNA)
• Strictly maternal transmission (mitochondria are maternally inherited)
• Heteroplasmy: cells contain a mixture of normal and mutant mitochondria - variable expressivity
• Threshold effect: disease manifests only when mutant mtDNA exceeds a threshold proportion
• Examples: MELAS (mitochondrial encephalomyopathy, lactic acidosis, stroke-like episodes), MERRF, Leber hereditary optic neuropathy (LHON)
• Tissues most affected: those with highest energy demands (neurons, muscle, retina)

3. Genomic Imprinting

• Epigenetic phenomenon where the expression of a gene depends on whether it was inherited from the mother or father
• Imprinted genes are "stamped" with the parental sex of origin during gametogenesis (DNA methylation)
• Example: Prader-Willi syndrome (loss of paternal 15q11-13 expression) vs. Angelman syndrome (loss of maternal 15q11-13 expression) - same chromosomal region, different clinical phenotypes depending on parent of origin

Important Concepts

---