PART 1 From “Mendel” to “Molecules”

Birth of Genetics

Genotype determining phenotype, phenotype deducing genotype
Trait/Character, Variant, Mutants

Single-Gene Inheritance

The Law of Segregation

Alleles: Genes(described as “Particle” by Mendel) exist in different versions called alleles (e.g., an allele for purple flowers and an allele for white flowers).
Separation: Diploid organism has two alleles, while only one in germs.
Random Assortment into Gametes: The separation is random—each allele has an equal chance of ending up in a given gamete.
Dominance and Recessiveness: If the two alleles an organism inherits are different (heterozygous), one allele (dominant) may mask the expression of the other (recessive). The recessive trait only appears if an individual inherits two copies of the recessive allele .
In modern perspective, we know that in a diploid organism (with two sets of chromosomes), the two alleles for a gene are located on a pair of homologous chromosomes. During meiosis, when gametes are produced, these homologous chromosomes separate. Consequently, each gamete ends up with only one chromosome from the pair, and therefore, only one allele for the gene.

Allele

Null allele: no function
Hypomorphic allele: weak function
Hypermorphic allele: over-functioned (overexpressed)
Usually, Null and hypomorphic alleles are recessive

Molecular Mechanisms for Dominance

Haploid insufficiency :mutation allele encodes an inactive protein; or, null allele;cells need high minimum level of the protein (e.g., >1/2 wt)
“Spoiler” alleles: mutated protein interferes with the normal protein (e.g., in dimer)
Gain-of-function alleles: mutated protein has a new activity that causes the phenotype(e.g., kinase no longer responds to negative regulation)

Sex-Linked Single-gene Inheritance

Different reciprocal crosses(正反交)
Not necessarily related to sex determination
eye color of Drosophila

Inheritance Patterns

Autosomal Recessive: Often inbreeding is implicated.
Autosomal Dominant: Affected cases appear in every generation. De novo mutation in 1st generation is possible
X-linked recessive disorders
X-linked dominant disorders
Y-linked disorders

Two-Gene Inheritance

Law of Independent Assortment

During gamete formation, the alleles of one gene segregate into gametes independently of the alleles of another gene, assuming the genes are located on different chromosomes (or are far apart on the same chromosome).

Working with Independent Assortment

Predicting the progeny ratio ：The Product Rule & The Sum Rule
How many progenies are needed? If one genotype has the probability of 1/256 to appear, can I find at least one targeted progeny from 256 total? ($\alpha=0.95$)
Does the real data fit the expectation based on a hypothesis? Chi-square test: $\chi ^2 = \sum \frac{(O-E)^2}{O}$
Synthesizing pure lines with combined traits:

Genetic Interaction

Genes almost never act alone, but as a pathway
Types of Interaction:
1. Negative Interaction:sick + sick = sicker/death
2. Positive Interaction: Mask( one mutation masks the expression of the other ) or Suppression (reduce the defect or reverse it to normal)

Recombination

Morgan: Chromosomes are the inheritance carrier; Genes are the inheritance unit; Genes are the recombination unit; Genes are linearly arranged on chromosomes
Chiasmata(染色体交叉点)：
1. DNA double strand breaks（DSB） are generated throughout the genome (mostly via specific enzyme, Spo11; rarely, via non specific DNA damage or replication stress)
2. Probability roughly correlates with the DNA fragment length (distance between two spots)
3. There are specific “hot spots” or “cold spots” for recombination in genome
4. At least one chiasma per pair of homologous chromosomes is required for proper segregation, often more are present
Genetic mapping:
1. 1 m.u.= 1% RF (recombination frequency)
2. relative order and distance, not absolute
3. Measured RF is smaller than the calculated value because of double or more crossovers(lead to non-recombination)

Molecular Basis of Genetics

Finding of DNA as the genetic material

Griffith (1928):
1. S strain -> dead;
2. R strain -> live;
3. S strain heat-killed -> live;
4. R strain + S strain heat-killed -> dead
Avery(1944): S strain extract , destroyed different components, only DNA destroyed group lived.
Hershey-Chase(1952): T2-Phage, S35 labeled protein and P32 labeled DNA.
Chargaff’s Rule: A=T, C=G
Franklin, Watson, Crick(1952): Double helix

Prokaryote/Phage (virus) Genetics

Bacteria Conjugation

F factor transfers from donor (F+) to receipient (F-) via “rolling circle” replication
Transfering is restricted to F factor
Integration of F factor into the genome converts F+ cell to hfr cell ( integrating spontaneously at random site)
hfr cell transfers part or all of its genome to F- via conjugation

Imprecise excision of integrated F generates F’ cells ：carrying another gene; useful for dominance test complementation, genetic interaction ……

Lac Operon

Lac operon is transcribed only at the presence of lactose or IPTG
Pajamo Experiment:
1. act at the DNA level
2. LacI acts as a repressor
3. O is cis-acting; LacI is trans-acting
Diauxic (diphasic) growth : use glucose first, lag phase, then use lactose
- catabolite repression :when glucose is high, cAMP is low. CAP cannot bind to the lac promoter, little mRNA. While glucose running out , cAMP is high. CAP-cAMP activates transcription of lac mRNA.

$\lambda$ Bacteriophage

Lytic(溶菌周期) or Lysogenic(溶源周期)
$\lambda$ repressor -> lytic; Cro -> lysogenic

Chromosome

Nucleosome

Histone
Not evenly distributed along chromosome, NFR are easily accessible(nucleosome free region)
Histone modification: Acetylation, Writer(HAT)&Eraser(HDAC). Methylation, Ubiquitylation, Phosphorylation …….
Histone Code: Different combinations of histone modifications create unique binding sites that can be read by transcription coregulators, thereby modulating transcription On and Off
Chromatin remodeling: In addition to modify histones in nucleosomes in situ, nucleosomes with chromatin can also be remodeled via sliding(滑动), ejection(排出) or replacement(替换)
ChIP : To detect Protein and DNA interaction. Cross-link; Break into pieces; Add antibody and pull down; Reverse cross-link; Purify DNA or Protein for analysis
Epigenetic Inheritance:
1. a change in phenotype that is heritable but does not involve DNA mutation
2. cells with the same genotype could have different phenotypes that persist for many generations
3. e.g: X–Inactivation: Y-chr. lost most of the genes on X; thus, male (XY) and female (XX) have different dosages for most X-chr. genes. Human females randomly inactivate one X in somatic cells (Barr Body); Drosophila males up-regulates the X-chr. genes by ~ 2X ———- Dosage Compensation.

Centromere

Centromere guides the assembly of kinetochore(动粒) for microtubule binding
Centromeres are determined by unique Cenp-A nucleosome (a histone H3 variant)

Telomere

a special DNA+protein structure, protects the ends of the chromosomes. (T-loop) Without protection, the ends of chromosomes are exposed dsDNA breaks, which are high risk factor causing genome instability
Telomerase, a special DNA polymerase (reverse transcription), dedicated to telomere replication

Telomere shortening and aging:
- Stem cells have active telomerase and possess full-length telomeres
- Somatic cells inactivate telomerase and shorten the telomeres with sequential rounds of cell division, eventually lead to cell aging

Karyotype（核型）

the number and the morphological features of chromosomes in the cell nuclei of a species
Chromosome mutations： Translocation, Inversion, Deletion, Missing chromosome, Duplication, Extra chromosome
Copy number change of chromosome:
1. Euploid(整倍体 complete set): Bypassing chromosome segregation or Nuclear division (2n->4n)
2. Aneuploid (非整倍体individual chromosome): Monoploid (recessive mutations masked in diploid are exposed); Triploid (problem in meiotic homolog-pairing); Tetraploid
Karyotype changes occur frequently during evolution

[!NOTE]

Why is aneuploidy particularly detrimental?

Gene-dosage effect:

Compared to diploid (euploid), aneuploids have certain genes that are of lower (or higher) dosage, and are out of balance with the rest of the genome. Polyploids maintain gene balance, despite of higher dosage.

Thanks to sex chromosome compensation (X-inactivation), XO and XXY are viable, although with defects. Also the Trisomy 21, because Chr.21 is the shortest in human genome.

Balancer Chromosome: contains multiple inversions; when it is combined with the corresponding wild-type chromosome, there can be no viable crossover products. So a specific combination of alleles on chromosome can be always preserved.

PART 2-1 Omics

Why is genomics important?

Before genomics, we usually observe the phenotype first, then find the geneby genetic mapping. After genomics, we can delete/mutate genes directly and see what happens to the phenotype.
Comparative genomics.
Making it possible to study genomic elements other than coding genes. (Non-coding DNA)
Biodiversity and evolution: it’s very cool to know the entirety of the genome of an organism and ponder the meaning of life.
Precision Medicine

What is a genome?

Genome is the aggregate of DNA/RNA information for an organism.
Genome contains all the information necessary for an organism to replicate.
Genome is organized and highly packed, with high-level interactions between different parts of the genome.

How do we sequence a genome?

Sanger sequencing – 1st generation
NGS– 2nd generation, The actual reading step is similar to that of the sanger sequencing, except in massive scale
Nanopore-3rd generation, Read about to millions of base pairs, but with more errors

How to Assembly a Whole Genome?

Contig（重叠群） -> Pari-ends Read（配对末端读取） -> Scaffold（有序框架）
Lander-Waterman Equation:
- $C=\frac{L \times N}{G}$
- C: coverage
- L: read length (bp)
- N: number of reads
- G: haploid genome length
- If we sequence a E. coli genome, suppose we make a paired-end sequencing run with L = 300, N = 3,113,148, and G = 4,578,159. The estimated coverage C would be: $\approx 204$
Poisson Distribution:
- $P(X=x)=\frac{\lambda ^x e^{-\lambda}}{x!}$
- x: Number of times a genomic position is sequenced
- $\lambda$: sequencing coverage C
- 为了确保基因组99%的区域至少被覆盖1次（即覆盖度达到99%），我们需要多少平均深度？即1%的区域没有被测到，$P(0) ≤ 1\% = 0.01$,解得$λ ≥ -ln(0.01) ≈ 4.6$

The Application of Genomics

Phylogenetics
Comparative genomics: copy number variations (CNV)
Genomic surveillance (基因组监测)

Functional Omics

ATAC-seq: Tn5 切割-> 加上测序接头 -> 测序，开放的染色质有比较高的峰
Hi-C ：detects chromatin interactions
Transcriptomics
Proteomics: Mass Spectrometry, LC/MS, can also identifying protein posttranslational modification
Metabolomics

PART 3-1 EvoDevo

Evolution: survival of the fittest vs. Evodevo: arrival of the fittest
EvoDevo: compare genes over a specific window of developmental process between species to infer how such process evolved

What is evolution?

Change of heritable traits of biological populations over successive generations

Why do we study evolution?

Reconstruct the history of gene, genomes, species
Explain the cause of the change, learn the general principle and exceptions of the tremendous biodiversity
Offer new ideas to other biology disciplines

What fuels evolution? Mutations!

Genome Duplications (Polypolidy): evidence—HOX gene
Chromosome fusion/fission/translocation/inversion：猫叫综合征
Gene Duplication：转座子
Substitutions & Indels ：镰状细胞贫血（碱基替换）、苯丙酮尿症（3个碱基缺失）、Tay-Sachs病（1个碱基插入，严重移码）
Alternative Splicing
RNA-editing

How does evolution work?

Natural selection acts on a POPULATION (not an individual) with variations
Variation -> Fitter ones survive -> Change of allele frequency
Sexual Selection may work in the opposite direction of natural selection

What is evodevo？

Deep homology: Sharing of the genetic regulatory apparatus that is used to build morphologically and phylogenetically disparate animal features (HOX gene and sex-determination gene DMRT1)
Toolkit genes: Genes or regulatory elements show deep conservation and control development of body parts and body building plan(Pax6 for eye development)
Modularity: Development occurs through a series of discrete and interacting modules: morphogenic fields, signaling pathways, imaginal disk, cell lineages
Heterotopy（location）, Heterochrony（time）, Heterometry（amount）, Heterotypy（kind）
Hourglass model: mid-embryonic represent the period of highest conservation

[!IMPORTANT]

发育与进化的区别？两者的联系？

区别：Linear Thinking vs. Tree Thinking

联系：EvoDevo 演化发育生物学

发育为演化研究提供素材（深层同源性、工具箱基因、异时发生与异位发生、发育过程本身限制了进化可能的方向）

PART 3-2 Evolution of Sex

What is sex?

Genetic change between individuals
Benefit of sex:
1. DNA repair
2. Sex produces genetic variation that can be exposed to selection
3. Sex can make selection process more effective
Disadvantages of sex:
1. 2-fold cost of sex
2. Producing the males, finding the mate, male-male competition
3. Predation risk
4. Disease spread
5. Recombination load: break the combination of beneficial alleles

How is sex determined?

XY (most mammals, Drosophila), ZW (snake, bird, butterfly, silkworm) and UV (algae) of sex chromosomes
Environmental sex determination: bird, turtle, crocodile
==Masters change, slave remain==: whatever sex chromosome changes, Dsx/Dmrt1 gene controls sex. (ON-MALE, OFF-FEMALE, or in different isoform)
Examples:
1. Drosophila: X:A<=0.5, Male. Sxl Alternative Splicing
2. Silkworm: ZW, Female
3. Human: Sry, Sox9, Wnt4
4. Red-earred slider turtle: High temperature, Female

How do sex chromosomes evolve?

[!IMPORTANT]

Y染色体的退化

性别决定基因在常染色体上发生，Y形成；

性别拮抗基因（sexually antagonistic gene）在Y染色体上积累：在一个性别中是有益的，而在另一个性别中却是有害的

为防止性别拮抗基因通过重组跳到另一染色体上对另一性别产生伤害，Y染色体局部发生==重组抑制==，该区域失去了通过重组“净化”有害突变的能力

重组抑制区域积累突变（转座子、无义突变、缺失、插入、重复序列），大量基因丢失或功能缺陷

X染色体的剂量补偿效应开始出现

Y染色体持续退化，最终可能消失

The complex Y-linked sequences
1. Dosage sensitive genes：退化得非常慢，一旦丢失大概率致死
2. Palindrome genes：Y染色体上的多拷贝基因，能延缓退化、修复某个拷贝的突变

How do dosage compensation evolve?

Drosophila: 2-fold upregulation in male
Nematode: 50% dampening in female
Mammal: one X inactive in femal (Barr Body)
Evolving within 1 million years

What are the remaining questions?

How do turnovers of sex chromosome systems happen？当Y染色体彻底消失后，原有的性染色体被新的性染色体所取代，从而形成新的性别决定机制性冲突
Why do some sex chromosomes never degenerate?
How do species determine their sex? 仍有很多物种的性别决定机制是未知的

PART 3-3 Are “Junk DNA” Junk?

Transportable Elements (TE)

Class I: Retrotransposons (反转录转座子) Copy and Paste
Class II: DNA Transposons (DNA转座子) Cut/Peel and Paste

The Deleterious Effects:
1. DNA Damage and Interrupt Genes
2. Genomic Rearrangement
3. Gene Silencing (TE affects chromatin accessibility，异染色质扩散)
4. Change Splicing Pattern（新剪切位点）
de novo TE insertions and human disease:
- Exon disruption : Haemophilia(血友病)
- Change of alternative splicing: Dent’s Disease

How TEs Contribute to the Genome Organization？

Act as genes—flamenco: piRNA, lncRNA
Form centromeres and telomeres
Participate in 3D Genome Organization:
- TE demarcate the TAD boundaries(划分拓扑关联域边界)
- Insertion of HERV-H retrotransposon creates a new TAD border

How TEs Regulate Gene Expression?

Providing TF binding sites: 人类催乳素增强子来源于一种DNA转座子
Provide insulator/boundary elements: 绝缘子、边界元件
Change Splicing Pattern
Transposase-TF fusion: create new TF
Tissue- or cell-specific expression patterns
Tissue- or stage-specific histone modifications

[!IMPORTANT]

转座子与编码蛋白质的基因的区别

PART 3-4 Mechanisms of Transcriptional Regulation

Histone code and chromatin state

Chromatin architecture

Important technology: Hi-C. Crosslink -> Digestion -> Biotin Marking -> Loop Ligation -> Break crosslink -> Sequencing Adaptor -> PCR -> Sequencing
Nucleosome
TAD: A topologically associating domain is a self-interacting genomic region, meaning that DNA sequences within a TAD physically interact with each other more frequently than with sequences outside the TAD
Chromatin compartments
1. A-compartments: internal regions, actively transcribed genes
2. B-compartments: the periphery of nuclei , inactive genes

Genome Folding

Loop Extrusion (环挤压): loops are produced by progressive extrusion of chromatin by a loop extrusion factor(like cohesin)，forming TAD
Self-interactions/Phase Separation(自相互作用/相分离): forming A/B compartment
1. attraction of heterochromatin to the nuclear lamina
2. preferential attraction of similar chromatin to each other
3. higher levels of chromatin mobility in active chromatin
4. transcription-related clustering of euchromatin.

TADs and Transcription

Enhancers and promoters can interact through chromatin looping, with TAD facilitating this interaction.

PART 3-5 Human Evolution

Human origin and migration history

Single Origin Hypothesis: out of Africa, displacing other archaic hominins
Multiregional: spread out of Africa much earlier, interbreeding with other archaic hominins (gene flow)
Neanderthals: 尼安德特人
Denisova: 丹尼索瓦人

Human unique traits

Intelligence and Language: non-coding DNA, FOXP2
High Altitude Adaptation: EPAS1

PART 4 人类遗传病

人类基因组组成

单拷贝DNA：绝大多数蛋白质编码基因
重复DNA：
1. 成簇重复序列（卫星DNA）：短序列串联重复，不同卫星DNA的重复核心也不一样
2. 散在重复序列：短散在重复序列（SINE）如Alu家族，约300bp；长散在重复序列（LINE），可以达到6kbp
3. 区段重复：高保守，在染色体上位置较远

染色体疾病

异常的染色体分离：非整倍体、单亲二体
频发性染色体综合征：在片段重复区域的重组
特发性染色体畸变：常染色体缺失（猫叫综合征）
性染色体畸变：Turner综合征（XO），Klinefelter综合征（XXY）

单基因遗传病

常染色体显性：通常患者的基因型均杂合子，患病概率高，代代出现
常染色体隐性：纯合致病，携带者不致病，近亲结婚容易出现
X连锁显性：无男-男遗传现象
X连锁隐性：通常仅见于男性，或男性患者远多于女性，女性多为携带者（血友病A、色盲）
假常染色体遗传：位于性染色体的假常染色体区域，和常显类似
三核苷酸不稳定重复扩增：亨廷顿舞蹈症等神经退行疾病
mtDNA母系遗传：患病父系与正常母系。后代突变基因丢失，纯质性与杂质性

人类遗传病分析方法

基于家系的连锁分析

利用家系追踪疾病在家族成员间的遗传，并检测疾病与特定基因组区域甚至特定突变的共遗传
重组：非常近完全连锁；非常远自由组合
连锁不平衡LD：分属两个或两个以上基因座的等位基因同时出现在一条染色体上的几率，高于随机出现的频率，说明两个基因在物理位置上非常近
LD Block: 具有高LD等位基因的位点簇，在人群中不同；LD块之间的边界通常与减数分裂重组热点相吻合
$LOD = log_{10}\frac{L(\theta)}{L(\theta = 0.5)}$
- 带入$\theta$从0-0.5，计算LOD值，最大的LOG>3时，此时的$\theta$ 就是重组率
例子：Family1 如下，可以发现F3中有一个AD不患病，一定是发生了重组，假设$\theta=0.1$计算得到，LOD=1.09。同时有其他几个家族，同样计算，计算LOD之和，发现0.06时候LOD大于3，即$\theta=0.06$

基于人群的全基因组关联分析（GWAS）

通过与同一人群中未受影响的对照组进行比较，发现受影响人群队列中特定等位基因频率的增加或减少
适用于非孟德尔遗传的复杂和多因素疾病,利于发现微小影响导致复杂特征的变异

病例对照研究：$OR=\frac{a/b}{c/d}$(不等于1的时候和标记有关联)
横断面或队列研究：$RR=\frac{a/(a+b)}{c/(c+d)}$
局限性：
1. 由人口分层引起的完全人为关联：群体中存在多个亚群（人种、宗教），个体只在亚群内交配
2. 统计显著性的临界值宽松易出现假阳性结果：GWAS一般需要$P<5\times 10^{-8}$
3. 往往不能直接发现致病基因，只是找到了和易感因素存在连锁不平衡的遗传标记

基于个体全基因组测序分析

适用于罕见的、没有足够的家系材料进行连锁分析的孟德尔疾病
通常需要筛选数据：
1. 保留蛋白质编码序列：全外显子测序
2. 丢弃高于罕见病预期的常见变异
3. 保留无义、移码、高保守剪接位点突变，丢弃同义突变或内含子突变
4. 与可能的遗传模式一致的突变
局限性：
1. 甲基化疾病、单亲二体疾病无法分析
2. 非编码区和调控区的变异无法解释
3. 高度重复预测无法检测