Mapping Mendelian Disease (Genomics)

Recombination creates genetic diversity by shuffling alleles between homologous chromosomes.

It ensures new combinations of traits in offspring, which is vital for evolution.

Helps repair DNA damage and maintain chromosome integrity.

An allele is a variant form of a gene at a specific locus on a chromosome.

Different alleles can result in variations in traits, such as eye colour or blood type.

No, they may be separated during recombination in meiosis.

The likelihood depends on their distance on the chromosome—closer genes are less likely to recombine.

Not always; linkage depends on their proximity.

Recombination events can separate linked alleles if they are far enough apart on the chromosome.

A network of thin, vein-like vessels spread throughout the body.

Includes lymph nodes, small bean-shaped structures located along these vessels.

Contains organs like the spleen, thymus, and tonsils that support immune function.

Maintains fluid balance by collecting excess fluid and returning it to the bloodstream.

Plays a key role in immune defense by transporting white blood cells and filtering pathogens.

Absorbs fats and fat-soluble vitamins from the digestive system via the lacteals.

A condition caused by the buildup of lymph fluid, leading to swelling, often in the arms or legs.

Results from damage or obstruction in the lymphatic system, such as from surgery or infection.

Compression therapy: Using bandages or garments to improve fluid drainage.

Manual lymphatic drainage: A specialized massage technique to encourage fluid movement.

Exercise: Promotes circulation and lymphatic flow.

In severe cases, surgery may be performed to remove excess tissue or improve lymphatic drainage.

They are often overlooked compared to the blood circulatory system but play an integral role in health.

Fluid homeostasis: Maintains balance by returning excess interstitial fluid to the bloodstream.

Immune function: Filters pathogens and supports immune responses.

Fatty acid transport: Absorbs fats and fat-soluble vitamins from the digestive tract.

They form an interweaving network in the capillary bed, working closely with blood vessels.

Chronic oedema caused by a developmental abnormality or dysfunction of the lymphatic system.

Often progressive, with phenotypes varying by age of onset, site, inheritance patterns, associated features, and genetic causes.

Leads to debilitating, embarrassing, and stressful symptoms with recurrent infections.

There is currently no medical cure for lymphoedema.

Research bridges the gap between laboratory findings ("bench") and practical patient treatments ("bedside").

By investigating genetic causes, inheritance patterns, and phenotypes using modern genetic and molecular techniques.

Gene identification by gene mapping:

Homozygosity mapping

Linkage analysis

GWAS (Genome-Wide Association Studies)

Finding disease-causing mutations:

Sequencing

Proving causation:

Using in silico, in vitro, and in vivo tools.

The tendency for alleles at neighbouring loci (e.g., A1, A2, B1, and B2) to segregate together during meiosis.

To be linked, two loci must lie very close together on the chromosome.

Linked loci are less likely to be separated by recombination during meiosis.

A haplotype defines multiple alleles at linked loci, marking chromosomal segments.

These segments can be tracked through pedigrees and populations to study inheritance patterns.

Recombination (cross-overs) during meiosis is more likely between loci separated by a greater distance.

Closely linked loci are less likely to be separated, maintaining their association across generations.

It helps identify loci associated with diseases by tracking how linked alleles segregate together in pedigrees and populations.

If an allele is linked to a disease locus, affected relatives will inherit the same allele more often than expected by chance.

This linkage helps trace the disease locus through families by identifying shared haplotype blocks.

Affected individuals in a family are more likely to inherit the same haplotype block (e.g., M3 – M4) as the disease locus.

This shared inheritance pattern indicates the proximity of the alleles to the disease locus.

Affected individuals in a family are less likely to inherit the same marker alleles (e.g., M5 – M8).

This suggests that the alleles and the disease locus are located far apart or on different chromosomes.

Haplotype blocks mark chromosomal regions and can indicate the genomic location of the disease-causing gene.

A gene mapping tool used to identify the genomic regions linked to diseases.

It involves using observed loci (alleles) to infer the location of unobserved loci (such as disease genes).

Family-based design, ranging from using a few large families to many small nuclear families or sib-pairs.

The primary goal is to find the genomic region(s) that are linked to a disease.

Traditional linkage analysis and Sanger sequencing are used to identify the cause of autosomal recessive primary lymphoedema.

These methods help pinpoint the genetic loci linked to the disease and identify the mutations responsible for the condition.

Primary lymphoedema often follows an autosomal recessive inheritance pattern.

Generalized lymphatic dysplasia refers to a condition characterized by abnormal lymphatic development, leading to fluid buildup and edema in various parts of the body.

Antenatal hydrops with ascites and pleural effusions

Oedematous at birth

Intestinal lymphangiectasia

Peripheral lymphoedema affecting the arms, legs, and face

Mild developmental delay

Step 1: Take a pedigree and collect as many DNA samples as possible.

Step 2: Use a tool to observe alleles and generate genotyping data for the pedigree.

Step 3: Generate a file with the pedigree information along with genotyping data from the SNP array.

Step 4: Run the file in a linkage program (e.g., Merlin or PLINK).

NPL stands for Nonparametric Linkage.

NPL provides a plot for each chromosome to assess linkage.

NPL does not require assumptions about the inheritance model (no need to specify a genetic model or pedigree structure), unlike parametric linkage testing.

NPL does not impose any rules on the inheritance pattern, meaning it does not require assumptions about the genetic model.

NPL focuses on detecting linkage by observing allele sharing between affected individuals without needing a predefined inheritance model.

NPL assesses genetic linkage by comparing allele patterns across families and identifying whether certain alleles are shared more frequently in affected individuals.

Parametric analysis imposes rules about inheritance patterns and disease frequency when assessing genetic linkage.

In autosomal dominant inheritance, affected family members are assumed to be heterozygous for the disease-causing allele (e.g., AB).

In autosomal recessive inheritance, affected family members are assumed to be homozygous for the mutant allele (e.g., AA).

Parametric analysis helps refine linkage results by taking into account specific inheritance patterns and disease allele frequencies, improving the accuracy of mapping disease-causing genes.

The result is displayed for each of the 23 chromosomes.

Parametric tests highlight regions with high LOD scores, indicating areas where affected individuals show a distinct inheritance pattern compared to unaffected individuals.

High LOD scores indicate regions where the affected individuals' genotypes follow the imposed inheritance pattern, and differ from unaffected individuals.

These regions are potential candidates for being linked to a disease-causing gene.

Parametric analysis helps by comparing the genotypes of affected vs. unaffected individuals, ensuring that the genotyping follows the expected inheritance pattern for the disease under study.

A LOD score > 3.0 is considered significant evidence for linkage, meaning the genetic locus is likely linked to the disease.

A LOD score > 3 corresponds to a p-value of approximately 0.05, which indicates statistical significance in the association between the genetic locus and the disease.

LOD scores between -2 and 3 are considered inconclusive, as they do not provide strong evidence for or against linkage.

LOD scores below -2 suggest significant non-linkage, meaning the genetic locus is unlikely to be associated with the disease.

Even though a LODmax of 2.1 is not significant, the two regions with LOD scores between -2 and 2.1 are worth exploring further because they were the only two regions above LOD score -2. This suggests they may be linked to the disease, but more investigation is needed.

These regions are considered for further exploration because they may still have a genetic association with the disease, especially as they were the only regions that exceeded the -2 LOD score threshold when applying parametric analysis for autosomal recessive inheritance.

Gene identification by gene mapping: This step involves identifying regions of the genome that are linked to a disease through methods like linkage analysis and GWAS.

Sequencing: This technique is used to analyze the DNA of candidate genes to find mutations that could potentially cause the disease.

In silico tools: These involve computer-based predictions, such as computational modeling, to assess the potential impact of mutations.

In vitro tools: These are laboratory-based techniques, such as cell culture studies, to observe the effects of mutations in a controlled environment.

In vivo tools: These refer to animal models or clinical studies where mutations are studied in living organisms to observe their effects on disease development.

Sanger sequencing: This method is used to sequence the DNA of the candidate genes to identify specific mutations that may be causing the disease.

Sanger sequencing: A method of DNA sequencing that uses selective incorporation of chain-terminating dideoxynucleotides.

Process: The gene of interest is amplified by PCR, and the sequence is read using fluorescent markers that indicate which base (A, T, C, G) is incorporated.

Identification of mutations: Any variations in the normal DNA sequence (mutations) can be detected by comparing the obtained sequence to a reference genome.

High accuracy: Sanger sequencing provides high-quality, accurate sequences for small regions of DNA, allowing precise identification of mutations.

Targeted sequencing: It allows for focused analysis of specific genes or regions within the genome, such as the 130 candidate genes, making it ideal for finding rare or known mutations associated with diseases.

Using in silico, in vitro, and in vivo tools: These methods are used to validate that a mutation in a gene is actually responsible for the disease.

In silico tools: These are computational methods used to predict the effect of genetic mutations on protein function and structure.

Role in proving causality: In silico tools can analyze the mutation's potential to alter the gene’s function or lead to disease by comparing it to known mutation databases and using predictive models.

In vitro tools: Laboratory-based experiments (such as cell cultures or protein assays) used to test the effects of the mutation on gene expression or protein function.

Validation: For example, scientists might introduce the mutation into a cell culture model to observe if the mutation disrupts the normal cellular function, suggesting a causal relationship to disease.

In vivo tools: Experiments conducted in living organisms, such as animal models (e.g., mice), that carry the mutation of interest.

Validation: By observing how the mutation affects the organism's phenotype or health, researchers can determine if the mutation leads to the disease, confirming its causality.

Autosomal dominant: This form is inherited in an autosomal dominant pattern, meaning one copy of the mutated gene is sufficient to cause the disease.

4-limb lymphoedema: Affects all four limbs, leading to swelling.

Pubertal/adult onset: The symptoms typically appear during puberty or in adulthood.

Associated with venous incompetence: There is often a co-occurrence of venous problems, such as venous insufficiency.

No other abnormalities: The condition primarily affects the limbs without other major abnormalities.

Linkage analysis: This technique helps identify regions of the genome linked to the disease by studying genetic inheritance patterns in families. In primary lymphoedema, it helps to narrow down the possible genomic regions associated with the condition.

Next-generation sequencing: This advanced method allows for high-throughput, detailed analysis of the genome, helping to pinpoint mutations in genes that might be responsible for the disease.

Gene identification: By sequencing the candidate genes, researchers can directly identify the mutation causing the autosomal dominant form of primary lymphoedema.

Collect DNA samples: Obtain DNA samples from family members, especially affected individuals, and unaffected controls.

Genotyping: Use a genotyping tool, like a SNP array or microarray, to analyze the DNA and generate data on genetic variants (SNPs) across the genome. The data will include information about the alleles present at specific loci for each family member.

Create a file with pedigree and genotyping data: Combine the pedigree information (family tree showing relationships and affected status) with the genotyping data to create a file for analysis.

Choose a linkage program: Use software such as Merlin or PLINK to run the linkage analysis.

Set inheritance model: In the software, set the inheritance pattern to autosomal dominant. This will assume that one copy of the mutant allele is sufficient to cause the disease. The program will then compare the genotypes between affected and unaffected family members to identify linked genomic regions.

Identifying linked regions: The analysis will identify regions of the genome that show consistent inheritance of certain alleles (haplotypes) in affected family members. If a region is linked to the disease, affected individuals will more frequently inherit the same alleles at specific loci.

Traditional Sanger sequencing

Sequences individual genes to identify mutations.

Candidate gene screen

Screens known genes associated with the disease.

Next generation sequencing (NGS)

High-throughput technique for sequencing multiple genes or genomes at once.

Whole genome sequencing (WGS)

Sequences the entire genome, including coding and non-coding regions.

Whole exome sequencing (WES)

Focuses on sequencing only the exons (coding regions) of the genome.

High throughput: NGS can sequence multiple genes or even entire genomes at once, speeding up the process.

Comprehensive: It allows for the detection of a wide range of mutations, including small insertions, deletions, and rare variants that may be missed by traditional methods.

Use WGS when...: You need to examine not only the exons but also the regulatory regions, introns, and other non-coding areas of the genome that may be important for disease.

Use WES when...: You want to focus on the protein-coding regions of the genome, which are more likely to harbor mutations directly related to disease.

Mutations in a single gene

Genetic analysis can help identify the causative mutations.

Linkage analysis is an example of such a technique.

Understanding genetics and the tools applied is necessary.

The process can be complex due to factors like gene interactions, inheritance patterns, and technical limitations.