Author name: doctorsrevision@gmail.com

Core idea Disease begins when a genetic change alters a cellular function. In cancer, successive somatic and sometimes germline changes disable growth control, evade cell death, sustain replication, remodel tissue and enable invasion. The task for a doctor is to connect variant → molecular mechanism → cellular phenotype → clinical behaviour → treatment or prevention. Learning outcomes Distinguish germline from somatic disease variants; explain loss-of-function, gain-of-function, dominant-negative and haploinsufficient mechanisms; describe oncogenes, tumour-suppressor genes, apoptosis and DNA-repair pathways; recognise inherited cancer syndromes; and interpret molecular results for diagnosis, prognosis, targeted therapy and family counselling. 1. From variant to disease phenotype A variant can alter a protein’s quantity, structure, location, interaction or regulation. A missense substitution may change an active site; a nonsense variant can truncate a protein; a splice-site variant can remove an exon; a copy-number change alters dosage; and a regulatory variant can change when a gene is expressed. The same gene may cause different phenotypes depending on the allele, tissue, developmental stage and modifying factors. Not every variant causes disease. Interpretation combines population frequency, segregation, functional evidence, computational prediction, phenotype fit and previous clinical knowledge. A variant of uncertain significance should not be used alone for irreversible treatment or predictive testing. 2. Germline and somatic variation Feature Germline variant Somatic variant Where present Egg or sperm lineage and usually every cell. Acquired in a cell or clone during life; may be absent from blood. Inheritance May pass to children. Usually not inherited by children, though mosaic germline involvement is possible. Clinical implication Predisposition, inherited syndrome, congenital disease or pharmacogenetic effect. Tumour initiation, progression, treatment resistance or acquired disease. Testing Blood or saliva often represents the constitutional genome. Tumour tissue, circulating DNA or paired tumour-normal testing may be required. A germline predisposition is not the same as inevitable disease. Penetrance describes the proportion of people with a variant who develop the phenotype; expressivity describes the range of severity or features. A person may carry a high-risk allele and remain unaffected, while another develops cancer early. 3. Functional classes of disease variants Loss of function The gene product is absent or reduced. Recessive disease may require loss of both alleles, while haploinsufficiency occurs when one normal copy cannot provide enough product. Gain of function The altered protein is overactive, active in the wrong place or given a new activity. One allele may be sufficient, as in many oncogene activations. Dominant-negative The abnormal product interferes with the normal product, especially in multimeric proteins. The phenotype can be more severe than simple loss of one copy. Dosage or regulatory change Deletion, duplication, enhancer disruption or epigenetic silencing changes how much, where or when a gene is expressed. 4. Cancer as an evolutionary genetic disease Cancer is usually monoclonal at origin but becomes genetically heterogeneous. A founding cell acquires a growth advantage and expands; additional mutations create subclones that differ in proliferation, invasion, immune evasion and treatment sensitivity. Selection by hypoxia, immunity or therapy can favour resistant clones. Carcinogenesis is multistep. DNA damage, replication error, defective repair and epigenetic change accumulate over time. Chromosomal gains, losses, translocations, amplifications and deletions can alter many genes at once. Environmental exposures such as tobacco smoke, ultraviolet radiation, chronic inflammation and selected infections increase damage, but the clinical cancer reflects interaction between exposure, tissue context and host biology. 5. Four major gene groups in cancer Proto-oncogenes and oncogenes — the accelerator Proto-oncogenes normally promote appropriate growth and survival. Activating mutation, gene amplification, increased transcription or chromosomal translocation can convert them into oncogenes. Oncogene activation is generally a gain-of-function process; one altered allele can drive signalling. Examples include KRAS, MYC, ERBB2/HER2 and BCR::ABL1. Tumour-suppressor genes — the brake Tumour suppressors restrain cell-cycle entry, repair damage, maintain tissue architecture or trigger apoptosis. Their loss often follows the two-hit model: an inherited or early first hit reduces reserve, then a somatic second hit removes the remaining function. RB1, TP53, APC and PTEN illustrate different tumour-suppressor pathways. DNA-repair genes — genome maintenance Repair genes correct mismatches, double-strand breaks, crosslinks and ultraviolet damage. When repair is defective, mutation burden and chromosomal instability rise. BRCA1/2 deficiency impairs homologous recombination; mismatch-repair deficiency produces microsatellite instability and may cause Lynch syndrome. Apoptosis and senescence regulators Damaged cells should stop dividing, enter senescence or undergo programmed cell death. Altered p53 signalling, BCL-2 family balance, death receptors or telomere control allows abnormal cells to survive and continue dividing. 6. Key pathways and examples TP53 responds to DNA damage and other cellular stress by inducing cell-cycle arrest, repair or apoptosis. Loss of p53 permits replication of damaged DNA and is common across cancers. Germline TP53 predisposition causes Li-Fraumeni syndrome and a broad spectrum of early malignancies. RB–E2F control restrains the G1-to-S transition. Cyclin D–CDK activity phosphorylates RB, releasing E2F to activate DNA-synthesis genes. Disruption of RB, cyclins, CDKs or upstream signals removes this checkpoint. APC–WNT signalling normally regulates beta-catenin degradation. APC loss allows beta-catenin accumulation and transcription of proliferative genes, contributing to familial adenomatous polyposis and colorectal cancer. RAS–RAF–MEK–ERK transmits growth-factor signals. Activating RAS or pathway mutations can make the pathway constitutively active, so downstream growth continues without normal external stimulation. PI3K–AKT–mTOR integrates growth and nutrient signals. Hyperactivation supports survival, metabolism and protein synthesis and can contribute to resistance. Telomerase maintains telomeres in many cancers, allowing cells to escape the replicative limit that constrains most normal somatic cells. 7. Inherited cancer-predisposition syndromes Syndrome or gene Characteristic pattern Clinical reasoning BRCA1/BRCA2 Breast, ovarian, prostate and pancreatic cancer risk. Risk assessment guides surveillance, reproductive counselling and selected targeted therapy. Lynch syndrome: MLH1, MSH2, MSH6, PMS2, EPCAM Colorectal, endometrial and other mismatch-repair-associated cancers, often at younger ages. Tumour testing for mismatch repair or microsatellite instability can guide germline evaluation. APC familial adenomatous polyposis Numerous colorectal adenomas and high colorectal-cancer risk. Early specialist surveillance and family testing are essential. RB1 hereditary retinoblastoma Early retinoblastoma and second-tumour risk. Urgent ophthalmic and genetic evaluation; relatives may need assessment. TP53 Li-Fraumeni syndrome Multiple early cancers, including sarcoma, breast, brain and adrenal tumours. Surveillance and

Core idea Chromosomal abnormalities are changes in chromosome number or structure that alter gene dosage, gene disruption or genome stability. Their clinical effects range from early miscarriage to recognizable syndromes, infertility, developmental disability, congenital malformations and cancer. The phenotype depends on the chromosome involved, the size and location of the change, mosaicism, uniparental disomy and the genes affected. Learning outcomes Explain normal chromosome organisation and notation; distinguish numerical from structural abnormalities; describe nondisjunction, anaphase lag and breakage-rejoining; interpret karyotype, FISH, microarray and sequencing results; recognise major viable aneuploidies; and counsel patients about mosaicism, recurrence risk and diagnostic limits. 1. Normal chromosome organisation Human somatic cells normally contain 46 chromosomes: 22 pairs of autosomes and one pair of sex chromosomes. Each chromosome contains a short arm (p), a long arm (q), a centromere and telomeres. During the cell cycle, DNA replicates in S phase so each chromosome temporarily consists of two sister chromatids joined at the centromere. A karyotype describes chromosome number and visible structure, not every base-pair sequence. Genes are distributed along chromosomes and are sensitive to dosage. A deletion may remove one copy of several genes; a duplication may create three copies; a balanced translocation can preserve total DNA but disrupt a gene or alter its regulation. The clinical effect is therefore not predicted by size alone. 2. Numerical abnormalities Aneuploidy is gain or loss of one or more individual chromosomes, such as trisomy or monosomy. Polyploidy is an extra complete chromosome set, such as triploidy. Aneuploidy usually results from nondisjunction—the failure of homologous chromosomes or sister chromatids to separate—or anaphase lag, in which a chromosome is left behind and excluded from a daughter nucleus. Mechanism Cellular event Typical consequence Meiotic nondisjunction I Homologous chromosomes fail to separate. Gametes receive both homologues or none; after fertilisation, trisomy or monosomy may result. Meiotic nondisjunction II Sister chromatids fail to separate. Two abnormal and two normal gametes may be produced. Mitotic nondisjunction Separation fails after fertilisation. Mosaicism: two or more cell lines in one individual. Anaphase lag A chromosome fails to migrate into one daughter nucleus. Loss of a chromosome in a daughter cell, producing mosaic monosomy. Polyploidy Extra complete haploid set from dispermy or meiotic failure. Usually severe malformations and early pregnancy loss. 3. Structural abnormalities Structural abnormalities follow chromosome breakage with abnormal repair. A deletion removes a segment; a duplication repeats it; an inversion reverses a segment; a translocation moves material between chromosomes; and a ring chromosome forms when both ends break and rejoin. An insertion places a segment into another location. Isochromosomes contain two copies of one arm and loss of the other. A structural change may be balanced or unbalanced. Balanced carriers may be healthy but produce gametes with partial monosomy or trisomy, causing miscarriage or an affected child. An apparently balanced change can still disrupt a gene or alter a regulatory landscape, so a normal-looking phenotype does not always mean zero risk. Deletion Loss of genetic material. Terminal deletions involve an end; interstitial deletions occur between two breakpoints. Severity reflects dosage-sensitive genes and mosaicism. Duplication Extra genomic material may increase gene dosage and cause developmental or congenital phenotypes. Duplications can be tandem, inverted or inserted elsewhere. Inversion A segment reverses orientation. Pericentric inversions include the centromere; paracentric inversions do not. Carriers may be normal but have reproductive risk from abnormal recombinant chromosomes. Translocation Reciprocal exchange may be balanced; Robertsonian translocation joins long arms of acrocentric chromosomes and can predispose to translocation trisomy. 4. Mosaicism and chimerism Mosaicism means genetically different cell lines derived from one zygote. Post-zygotic nondisjunction or anaphase lag may produce a normal and an abnormal line. The percentage in blood may not represent the percentage in brain, heart, placenta or gonads, so a low blood mosaic result does not automatically predict a mild phenotype. Chimerism refers to cell lines from two different zygotes, for example after twin fusion or transplantation. Confined placental mosaicism occurs when abnormal cells are present in placenta but not the fetus; it can cause an abnormal screening result while fetal diagnostic testing is normal, or affect placental function and growth. Mosaicism requires context: report the tissue tested, cell count, method, percentage and detection limit. Never convert “20% mosaic in blood” directly into “20% affected body.” 5. Major viable aneuploidies Trisomy 21 (Down syndrome) Trisomy 21 most often results from meiotic nondisjunction, but translocation and mosaic forms occur. Typical findings may include characteristic facial appearance, hypotonia, developmental delay, congenital heart disease, hearing or vision problems, thyroid disease, gastrointestinal anomalies and increased risk of acute leukemia and Alzheimer-type pathology. Features vary widely; a karyotype and clinical assessment are needed rather than diagnosis by appearance alone. Trisomy 18 (Edwards syndrome) Trisomy 18 is associated with severe growth restriction, clenched hands with overlapping fingers, characteristic foot posture, congenital heart disease, brain and renal anomalies and high infant mortality. Mosaic or partial forms may be less severe. Care should include clear communication of prognosis, comfort, family goals and specialist review. Trisomy 13 (Patau syndrome) Trisomy 13 may cause holoprosencephaly, cleft lip or palate, microphthalmia, polydactyly, congenital heart and renal anomalies and severe neurodevelopmental impairment. The diagnosis should be confirmed and management individualised; counselling must avoid assuming that every child has the same course. 45,X and sex-chromosome aneuploidies Turner syndrome (45,X or mosaic variants) may present with short stature, gonadal dysgenesis, infertility, webbed neck, coarctation of the aorta, renal anomalies, hearing problems and specific learning difficulties. Klinefelter syndrome (47,XXY) commonly causes small firm testes, infertility, reduced androgenisation, tall stature and variable language or executive difficulties. 47,XXX and 47,XYY have variable, often subtle phenotypes. Explain that a sex-chromosome result does not predict personality or worth. 6. Microdeletions, duplications and copy-number change Some clinically important abnormalities are too small for routine karyotyping. Chromosomal microarray detects genome-wide gains and losses at higher resolution and can identify pathogenic copy-number variants in developmental delay, congenital anomalies, autism or multiple malformations. It may also reveal variants of uncertain significance or consanguinity-related regions of homozygosity, so pre-test counselling is essential. 7.

Core idea Sex determination establishes the developmental pathway of the gonads, internal ducts and external genitalia; sex differentiation is the subsequent process by which those tissues develop. Chromosomal sex, gonadal sex, hormonal environment and phenotypic sex are related but not identical. Disorders of sex development (DSD) occur when chromosomal, gonadal or anatomical development does not follow the usual pathway. Learning outcomes Explain chromosomal and molecular sex determination; trace SRY–SOX9 and ovarian pathways; compare X-linked, Y-linked, pseudoautosomal, sex-limited and sex-influenced inheritance; calculate recurrence risks; and approach a newborn or patient with atypical genital development safely and respectfully. 1. Chromosomal and gonadal foundations Most individuals with a 46,XX karyotype develop ovaries and most with 46,XY develop testes, but this is a probability rather than a complete definition of biological development. The Y chromosome may carry SRY, which initiates a testis pathway in the supporting cells of the undifferentiated gonad. In the absence of an effective testis pathway, ovarian development is supported by genes including WNT4, RSPO1 and FOXL2. Testis differentiation produces Sertoli cells, anti-Müllerian hormone and Leydig cells, which produce testosterone. Anti-Müllerian hormone causes Müllerian duct regression; testosterone supports Wolffian duct development; conversion of testosterone to dihydrotestosterone by 5-alpha-reductase supports prostate and external male genital development. Without these signals, Müllerian structures can develop and external genital development follows the female-typical pathway. 2. Sex-determining pathways and clinical disorders Pathway or condition Mechanism Clinical consequence SRY or SOX9 activation Supports testis development and Sertoli-cell differentiation. Loss or dysfunction may cause 46,XY gonadal dysgenesis; duplication or dysregulation may produce testicular development in an XX context. Androgen insensitivity 46,XY cells cannot respond fully to androgen through the androgen receptor. Complete forms may present with female external phenotype and absent uterus; partial forms have variable genital development. 5-alpha-reductase deficiency Reduced conversion of testosterone to dihydrotestosterone. Variable undervirilisation at birth with possible virilisation at puberty. Congenital adrenal hyperplasia Excess adrenal androgen in a 46,XX fetus, often from 21-hydroxylase deficiency. Variable virilisation, salt-wasting risk and need for urgent endocrine care. 45,X or mosaic Turner pattern Absent or abnormal second sex chromosome affects gonadal development. Short stature, ovarian insufficiency and cardiovascular or renal anomalies. 3. X-linked inheritance Genes on the X chromosome follow a characteristic pattern because males usually have one X chromosome while females have two. A male transmits his X chromosome to all daughters and his Y chromosome to all sons; therefore an X-linked trait cannot pass directly from father to son. A heterozygous carrier mother may transmit the variant to half of her sons and half of her daughters, although penetrance and X-inactivation can alter expression. X-linked recessive disorders include haemophilia A, haemophilia B, Duchenne muscular dystrophy and red–green colour vision deficiency. Affected males are often more severely affected because they lack a second allele. Females may be unaffected, mildly affected or clinically significant because of skewed X-inactivation, Turner karyotype, homozygosity or a variant with substantial effect. 4. X-linked dominant inheritance An X-linked dominant condition can affect females and males, sometimes with greater severity in males. An affected father transmits the variant to all daughters and no sons. An affected heterozygous mother has a 50% chance of transmitting it to each child. Some conditions are male-lethal, producing an apparent excess of affected females and recurrent miscarriages. 5. Y-linked inheritance Y-linked genes pass from father to son only. Affected fathers transmit the variant to all biological sons and to no daughters. Y-linked infertility genes in the AZF regions can be lost or deleted, and a man may pass the deletion to sons conceived with assisted reproduction. A Y-linked pattern requires strict male-to-male transmission and should not be assigned from a single affected father and son without molecular evidence. 6. Pseudoautosomal and sex-influenced traits Pseudoautosomal genes lie in regions shared by X and Y chromosomes and can recombine during male meiosis. Their inheritance resembles autosomal inheritance because both sexes carry a copy, but copy number and sex-chromosome context still matter. Sex-limited traits occur in only one sex because anatomy or hormones are required, while sex-influenced traits are expressed differently in males and females. Male-pattern baldness is a teaching example, but its biology is polygenic and should not be reduced to a single-gene rule. 7. Pedigree reasoning Ask whether affected males are related through mothers, whether father-to-son transmission exists, whether all daughters of an affected father are affected, and whether both sexes are affected. Record consanguinity, miscarriages, infertility, neonatal deaths and variable expression. The pedigree suggests an inheritance pattern; molecular testing confirms or refines it. Risk examples A carrier mother for an X-linked recessive disorder has a 25% chance of an affected child overall, or a 50% chance among sons. An affected father with an X-linked disorder transmits the variant to all daughters and no sons. An affected father with a true Y-linked variant transmits it to all biological sons. An affected heterozygous mother for an X-linked dominant condition has a 50% chance of transmission to each child. 8. Approach to atypical genital development Do not assign blame or rush a cosmetic procedure. Stabilise the newborn first, especially check for hypoglycaemia, dehydration, vomiting, shock and salt-wasting congenital adrenal hyperplasia. Take a pregnancy, family, medication and consanguinity history; examine carefully; obtain electrolytes, glucose, 17-hydroxyprogesterone and other endocrine tests as indicated; and request karyotype or rapid chromosome testing with specialist input. Use respectful language and protect privacy. A multidisciplinary team may include paediatrics, endocrinology, genetics, urology or gynaecology, psychology and ethics. Explain uncertainty honestly, avoid unnecessary photography or repeated examinations, and involve parents in decisions while considering the child’s future autonomy. 9. Clinical relevance of sex-linked disease When a patient has an X-linked disorder, identify carrier relatives, offer molecular confirmation, discuss reproductive options and plan condition-specific surveillance. In Duchenne muscular dystrophy, a maternal carrier may need cardiac assessment and genetic counselling; in haemophilia, invasive procedures and bleeding risk require coordinated haematology care. Inherited Y-chromosome deletions mainly affect fertility and may have implications for sons conceived through assisted reproduction. 10. Examination summary SRY initiates a testis pathway; Sertoli cells produce anti-Müllerian hormone

Core idea Inheritance describes how genetic information passes between generations and how genotype becomes phenotype. Mendelian ratios are models based on segregation and independent assortment; real patients often show incomplete penetrance, variable expressivity, mitochondrial transmission, imprinting, anticipation, polygenic effects or environmental modification. Learning outcomes Apply Mendel’s laws; construct Punnett squares and pedigrees; distinguish dominant, recessive, X-linked and mitochondrial patterns; explain incomplete dominance, codominance, allelic heterogeneity, epistasis, imprinting, anticipation, mosaicism and multifactorial inheritance; and communicate recurrence risk without treating a ratio as a diagnosis. 1. Mendel’s laws and the language of genetics An allele is an alternative form of a gene at a locus. A homozygote has two identical alleles; a heterozygote has two different alleles. The genotype is the allele combination, while the phenotype is the observed trait produced through gene expression, development and environment. A dominant allele is expressed in a heterozygote; dominance does not mean common, stronger or more normal. Segregation: the two alleles at a locus separate during meiosis so each gamete receives one. Independent assortment: alleles at different loci assort independently when loci are unlinked or far apart. Uniformity: crossing true-breeding parents can produce a uniform first-generation phenotype. Linkage, recombination and selection explain why real data may differ from simple ratios. 2. Mendelian inheritance patterns Pattern Pedigree clues Clinical examples Autosomal dominant Vertical transmission, both sexes, male-to-male transmission possible; affected heterozygote may have affected and unaffected children. Marfan syndrome, Huntington disease, familial hypercholesterolaemia. Autosomal recessive Affected siblings with unaffected parents, both sexes equally, consanguinity may be present. Sickle-cell disease, cystic fibrosis, phenylketonuria. X-linked recessive More affected males; no father-to-son transmission; carrier mothers may have affected sons. Haemophilia, Duchenne muscular dystrophy. X-linked dominant Affected father transmits to all daughters and no sons; affected mother transmits to half of children. Some forms of hypophosphataemic rickets. Y-linked Father-to-all-son transmission only. Selected Y-chromosome infertility deletions. 3. Probability and recurrence risk For two heterozygous autosomal carriers, each pregnancy has a 25% chance of an affected child, 50% chance of a carrier child and 25% chance of an unaffected non-carrier child. These are independent probabilities for each pregnancy, not a quota. The chance of two affected children in succession is 1/4 × 1/4 = 1/16; the chance of at least one affected child in three pregnancies is 1 − (3/4)^3. Use Bayes’ theorem when family history or a test changes the prior probability. The patient’s risk after testing depends on the sensitivity, specificity, likelihood ratio and starting risk. Never present a population risk as the individual’s risk without considering pedigree and phenotype. 4. Incomplete dominance and codominance In incomplete dominance, the heterozygote has an intermediate phenotype; this does not mean the alleles blend permanently. In codominance, both alleles are expressed. The ABO blood-group system is a clinical example: IA and IB are codominant, while i is recessive. A person with IAIB expresses A and B antigens. 5. Multiple alleles, lethal alleles and allelic heterogeneity A population may have many alleles at a locus even though each person carries only two. Lethal alleles can distort expected ratios because affected embryos may not survive to be counted. In allelic heterogeneity, different variants in the same gene produce the same disorder or different severities. In locus heterogeneity, variants in different genes produce a similar phenotype, making a single negative gene test insufficient. 6. Penetrance and variable expressivity Penetrance is the proportion of people with a genotype who express a phenotype. Incomplete penetrance can make a dominant condition appear to skip generations. Expressivity is the range of manifestations among people with the same genotype. Explain these concepts when a family member carries a pathogenic variant but appears healthy or mildly affected. 7. Pleiotropy and epistasis Pleiotropy occurs when one gene affects several organ systems; fibrillin-related Marfan syndrome can involve the skeleton, eyes and aorta. Epistasis occurs when one gene modifies or masks the effect of another. In humans, pathway interactions help explain why a variant does not create one isolated symptom but a network of findings. 8. Linkage and recombination Genes close together on the same chromosome tend to travel together. Crossing-over during meiosis creates recombinant chromosomes, and recombination frequency estimates genetic distance in centimorgans. Linkage analysis can track a disease allele through a family when the causal variant is unknown, but recombination and phenocopies limit certainty. 9. Mitochondrial inheritance Mitochondria are usually inherited through the oocyte, so an affected mother may transmit a mitochondrial variant to all children while an affected father generally does not transmit it. Heteroplasmy means a cell or person contains a mixture of normal and mutant mitochondrial genomes. The proportion and tissue distribution influence threshold effects, age of onset and severity. 10. Genomic imprinting and uniparental disomy Imprinting marks genes according to parental origin, usually through DNA methylation and chromatin changes. A deletion or uniparental disomy may cause disease only when the altered chromosome comes from a particular parent. Prader–Willi and Angelman syndromes illustrate parent-of-origin effects involving chromosome 15, but molecular testing must determine the mechanism. 11. Anticipation and repeat expansion In anticipation, a repeat expansion becomes larger or more unstable in successive generations, often causing earlier onset or greater severity. Huntington disease, myotonic dystrophy and fragile X-related disorders illustrate repeat dynamics, though the direction and parent-of-origin effect differ. A family history should record age at onset and exact molecular diagnosis rather than simply “runs in the family.” 12. Mosaicism and de novo variants Mosaicism results from a post-zygotic mutation, so different tissues may carry different cell lines. A blood test can be negative when the relevant tissue is affected. A de novo variant appears newly in the patient and is absent from parental blood, but parental mosaicism can make recurrence risk higher than the usual low estimate. 13. Polygenic and multifactorial inheritance Many common disorders result from several variants of small effect plus environment. Diabetes, hypertension, cleft lip and palate and neural-tube defects illustrate multifactorial patterns. Risk is often described as familial aggregation rather than a simple Mendelian percentage. Empiric recurrence data, family history, maternal health and environmental exposures

Core idea Molecular biology explains how information is stored in DNA, copied, repaired, expressed as RNA and protein, and regulated in cells. Clinical medicine depends on these processes: replication errors cause mutations, repair defects cause cancer susceptibility, transcription defects cause disease, and altered proteins change physiology. Learning outcomes Describe DNA structure and replication; explain polymerase fidelity and repair; compare transcription and RNA processing in prokaryotes and eukaryotes; trace translation and protein folding; and connect molecular defects to laboratory diagnosis and disease. 1. DNA structure and information storage DNA is a double helix of antiparallel polynucleotide strands. Each nucleotide contains deoxyribose, phosphate and a nitrogenous base. Adenine pairs with thymine through two hydrogen bonds; guanine pairs with cytosine through three. The sequence carries information, while the sugar-phosphate backbone provides chemical stability. DNA is packaged around histone proteins into nucleosomes and higher-order chromatin. Euchromatin is relatively accessible for transcription; heterochromatin is more condensed. Chromatin state can change without altering the DNA sequence through methylation, histone modification, nucleosome repositioning and non-coding RNA. 2. Semiconservative DNA replication Replication begins at origins where helicase separates the strands. Single-strand binding proteins stabilise the template; topoisomerases relieve torsional stress; primase lays RNA primers; and DNA polymerase extends a new strand only in the 5′→3′ direction. The leading strand is continuous, while the lagging strand is made as Okazaki fragments that are joined by ligase. Replication is accurate because polymerases select complementary bases, proofread newly added nucleotides and cooperate with post-replication repair. Telomeres protect chromosome ends, and telomerase extends telomeric repeats in germ cells, stem cells and many cancers. 3. DNA damage and repair Damage Repair pathway Clinical relevance Small base mismatch after replication Mismatch repair Defects cause microsatellite instability and Lynch syndrome. Bulky ultraviolet-induced lesion Nucleotide-excision repair Defects cause photosensitivity and xeroderma pigmentosum. Deaminated or oxidised base Base-excision repair Protects against endogenous metabolic damage. Double-strand break Homologous recombination or non-homologous end joining Repair failure causes chromosomal instability and cancer susceptibility. Interstrand crosslink Fanconi-associated repair networks Defects cause marrow failure, congenital anomalies and cancer risk. 4. Transcription: DNA to RNA RNA polymerase reads a DNA template and synthesises RNA 5′→3′. Promoters recruit transcription factors and polymerase; enhancers can act at a distance through chromatin looping. The primary transcript of a eukaryotic protein-coding gene receives a 5′ cap, introns are removed by spliceosomes and a 3′ poly-A tail is added. Alternative splicing allows one gene to produce different transcripts. Splicing errors can cause exon skipping, intron retention or abnormal reading frames. A molecular test should therefore consider whether the variant affects a splice enhancer, branch point or canonical donor/acceptor site. 5. RNA types and post-transcriptional control Messenger RNA carries coding information; transfer RNA brings amino acids; ribosomal RNA forms the catalytic core of ribosomes. MicroRNAs and long non-coding RNAs regulate stability, translation and chromatin. RNA editing and selective degradation alter the amount and form of transcript available for translation. 6. Translation and protein fate Ribosomes read mRNA codons from 5′ to 3′. Translation begins at a start codon, proceeds through peptide-bond formation and ends at a stop codon. Protein folding, disulfide-bond formation, glycosylation, phosphorylation, cleavage and trafficking determine whether the protein reaches the correct compartment and functions properly. A variant may produce a stable but inactive protein, a misfolded protein retained in the endoplasmic reticulum, a toxic aggregate, or a protein with abnormal location. These mechanisms explain why the same DNA-level category can produce different clinical phenotypes. 7. Mutations and molecular consequences Substitutions may be synonymous, missense, nonsense or affect splicing. Insertions and deletions can cause a frameshift; repeat expansions may become unstable; structural variants alter gene dosage or regulation. A mutation can be pathogenic through loss of function, gain of function, dominant-negative interference or dosage imbalance. 8. Laboratory applications PCR Amplifies a defined DNA region through denaturation, primer annealing and extension. Used for pathogen detection, genotyping and confirmation, but vulnerable to contamination and primer mismatch. Reverse-transcription PCR Converts RNA to cDNA before amplification and measures transcript abundance or splice products. Electrophoresis Separates nucleic acids by size and charge, allowing product-size comparison and fragment analysis. Sequencing Reads nucleotide order. Sanger sequencing is targeted; next-generation sequencing examines many genes but requires coverage, quality control and interpretation. 9. Molecular biology in clinical reasoning When selecting a test, start with the phenotype and suspected mechanism. A karyotype or microarray is more useful for chromosome-level change; a targeted PCR or Sanger test suits a known variant; a panel, exome or genome may be used for genetically heterogeneous disorders. A negative result means the method did not identify a reportable change; it does not exclude every molecular mechanism. Interpret the whole chain: DNA variant → RNA effect → protein effect → cellular pathway → phenotype. A laboratory report is not a diagnosis until the molecular finding fits the clinical picture and test limitations. 10. Examination summary DNA replication is semiconservative and 5′→3′; transcription produces RNA that is capped, spliced and polyadenylated; translation converts codons into protein; repair pathways maintain genome stability. Molecular disease may result from sequence, copy-number, regulatory, splicing or epigenetic change. PCR, electrophoresis and sequencing reveal different parts of the pathway and must be chosen according to the clinical question. References SlideShare: Basics of Molecular Biology. NCBI Bookshelf: Molecular Biology of the Cell. NHGRI Genetics Glossary.

The Genetic Code and Gene Regulation: How Cells Read, Control and Use DNA The genetic code is the rule that converts a nucleotide sequence in messenger RNA into an amino-acid sequence. Gene regulation determines when, where and how strongly that information is used. Together they explain how one genome produces many specialised cells and why abnormal regulation causes developmental disease, endocrine disorders, immune disease and cancer. Why this topic matters: a patient may have a normal gene sequence but disease because the gene is not switched on, is expressed in the wrong tissue, produces unstable RNA, translates inefficiently, or makes too much protein. 1. The central dogma and its limits In most human cells, information flows from DNA to RNA to protein. Transcription copies DNA information into RNA; translation reads mRNA codons to build a polypeptide. This is not a claim that every gene produces protein—some genes produce functional RNA—and it does not mean regulation occurs at only one step. Information flow DNA sequence → transcription initiation → pre-mRNA → 5′ capping, splicing and polyadenylation → mature mRNA → ribosome reading codons → polypeptide folding and modification → functional product and phenotype. 2. What the genetic code is The code is read in non-overlapping triplets called codons. Each codon contains three mRNA bases and specifies one amino acid or a stop signal. The code is degenerate because several codons can specify the same amino acid, but it is not ambiguous: a particular codon normally has one meaning in the standard code. AUG usually acts as the start codon and also encodes methionine. UAA, UAG and UGA are stop codons. The reading frame is established at initiation; shifting it changes all downstream codons. 3. Properties of the code Property Meaning Clinical relevance Triplet Three bases form one codon Insertions/deletions not divisible by three cause frameshifts. Degenerate Several codons may specify one amino acid Some substitutions are synonymous, but may still affect splicing or expression. Nearly universal Most organisms use the same code Allows recombinant DNA and gene-expression systems. Non-overlapping Each base is read in one codon within a frame A frameshift changes the downstream message. Wobble Pairing at the third codon position is flexible Explains redundancy and some effects of tRNA abundance. 4. Transcription: making the RNA message RNA polymerase II binds a regulated promoter and synthesises a complementary RNA strand using the DNA template. Transcription has initiation, elongation and termination. Transcription factors determine which genes are accessible and help recruit or block polymerase. Regulation at transcription is powerful because it controls whether a message is made at all. A hormone-responsive promoter, for example, can rapidly increase the production of enzymes or receptors after a signal reaches the nucleus. 5. RNA processing and quality control 5′ cap: protects the transcript and helps ribosome recognition. Splicing: removes introns and joins exons. Alternative splicing: produces different transcripts from one gene. Poly-A tail: supports stability, export and translation. Nonsense-mediated decay: removes selected transcripts with premature stop codons. Errors in processing can produce disease even when the coding sequence is unchanged. This is why variant interpretation must include splice-site, deep-intronic, UTR and regulatory regions. 6. Translation: decoding mRNA at the ribosome Translation begins when the ribosome identifies the mRNA start region and assembles around the start codon. Initiator tRNA carries methionine. During elongation, tRNAs deliver amino acids, peptide bonds form, and the ribosome moves codon by codon. A stop codon recruits release factors rather than a tRNA. Translation may occur on free ribosomes or ribosomes attached to the rough endoplasmic reticulum. The destination of the new protein depends on signal sequences and determines whether it becomes cytosolic, nuclear, mitochondrial, lysosomal, secreted or membrane-bound. 7. Levels of gene regulation Chromatin level DNA packaging controls accessibility to transcription factors. Transcriptional level Promoters, enhancers, repressors and transcription factors control RNA production. RNA-processing level Capping, splicing, editing and polyadenylation determine the mature message. RNA-stability level Degradation determines how long an mRNA remains available. Translational level Ribosome recruitment and initiation determine protein production. Post-translational level Folding, modification, trafficking and degradation determine protein activity. 8. Transcription factors and regulatory elements Transcription factors are DNA-binding proteins that activate or repress genes. Activators recruit co-activators, mediator and chromatin-opening machinery. Repressors recruit co-repressors and chromatin-condensing enzymes. The same gene can respond to several factors, allowing integration of hormones, nutrients, stress, inflammation and cell identity. 9. Epigenetic regulation Epigenetic mechanisms change gene activity without changing the underlying base sequence. DNA methylation can reduce promoter accessibility. Histone acetylation often opens chromatin, while other histone marks can activate or repress depending on their location and context. Epigenetic regulation is essential in embryonic development, X-inactivation, imprinting, ageing and cancer. Clinical caution: “epigenetic” does not mean that a disease is imaginary or that a patient caused it through lifestyle. Epigenetic states are influenced by development, environment, inflammation, nutrition and disease, but causal interpretation requires evidence. 10. Non-coding RNAs Ribosomal RNA forms the structural and catalytic core of ribosomes. Transfer RNA carries amino acids. MicroRNAs bind target mRNAs and reduce translation or promote degradation. Long non-coding RNAs can influence chromatin, transcription and RNA processing. Non-coding RNA abnormalities are increasingly recognised in cancer and inherited disease. 11. Operons and eukaryotic regulation Many bacteria organise related genes into operons controlled by one promoter and regulatory region. This allows coordinated response to nutrients. Human genes are usually regulated individually through combinations of promoters, enhancers, insulators and chromatin domains, although shared regulatory networks coordinate pathways. 12. Mutations in the code versus mutations in regulation Variant location Likely first effect Example clinical reasoning Coding exon Protein sequence or termination changes Assess missense mechanism, nonsense-mediated decay or domain effect. Promoter/enhancer Amount or tissue of expression changes Normal protein sequence may be made in the wrong quantity. Splice region Abnormal mature mRNA RNA studies may clarify exon skipping or intron retention. UTR Stability, localisation or translation changes Protein may be reduced despite a normal coding sequence. Epigenetic control Gene switched off or misregulated Consider imprinting, methylation disorders or tumour silencing. 13. Gene regulation in cancer Cancer