CLINICAL GENETICS
Clinical genetics are needed in the case of rare disorders (< 1:2000). In 80% of the cases, the rare disorder
is monogenetic. There is learned from the rare disease and precision medicine is applied. The basics in
clinical genetics are equal to the basics in personalized medicine. Major subjects in clinical genetics are:
Oncogenetics (hereditary breast or colon cancer) (41%)
Dysmorphology / syndromes (28%)
Prenatal diagnosis / counselling
Ultrasound abnormalities
Familial hereditary disorder
Chromosomal abnormalities
Cardiogenetics (HCM, ARVC, NCCM)
Connective tissue disorders (e.g. osteogenesis imperfecta)
Neurogenetics (M. Huntington, cerebellar ataxias)
Physical examination and diagnosis
When going to the clinical geneticist, a lot of different questions can be asked:
What is the diagnosis / syndrome?
o A person with complaints or the parents
Pregnancy related
o Congenital anomalies syndrome?
o Recurrence risk of familial disorders
o Chromosomal abnormalities (prenatal diagnosis)
Related to own health
o Hereditary disorders that occur later in life (Huntington)
o Increased risk for cancer (e.g. breast cancer, colon cancer)
o Increased risk for sudden cardiac death
Preventive medicine
Targeted therapy
Related to reproduction
o Consanguinity
o Increased risks based on ethnic background (e.g. Tay Sachs disease in Ashkenazi Jewish
people)
o Recurrence risk of familial disorders
Children are referred to a clinical geneticist when they suffer from mental retardation, multiple
congenital abnormalities, dysmorphism, single congenital abnormalities (cleft lip and/or palate), hearing
loss, congenital cataract, or abnormal growth (short stature or obesity).
What does a diagnosis add in children with MR/congenital abnormalities?
Answers on why relieve feelings of guilt
No need for investigations for other causes
Information on prognosis, treatment
Qualify for support, school options etc
Parental contact
Family members
Recurrence risk, reproductive choices
o Deciding not to have (more) children
o Accepting the risk
o Adoption
o Donor sperm or eggs
o Prenatal diagnosis (NIPT, CVS, AP), followed (or not) by termination of pregnancy
, o Pre-implementation genetic testing (PGT) Only use unaffected embryos
o Adapting partner choice (culturally related)
Clinical genetics is a broad specialism. Family history and physical examination are important for
diagnosis. Counselling before pregnancy is recommended. Clinical genetics provide information on which
counselees can base their own decisions. A genetic diagnosis may enable targeted therapy.
Diagnostic testing
PCR is the exponential amplification of a specific target piece of DNA.
There is a three step synthesis reaction:
1. Desaturation of template to single strands
2. Annealing of primers to each original strand (complementary
binding of the primers to the template)
3. Extension of the new DNA strands from the primers onwards
Needed for PCR are Taq polymerase, primers (forward and reverse),
template DNA, and nucleotides (dNTPs).
In Sanger sequencing, DNA sequencing is used to determine the precise order of the nucleotides of a
given DNA fragment. The reaction mixture consists of primer, DNA-template, DNA pol, dNTPs and ddNTPs
with fluorochromes. The mechanism works as follows:
1. DNA-chain termination via PCR
DNA is copied using special building blocks
Some stop the DNA from growing further
Creates many DNA pieces of different lengths
2. Size separation by gel electrophoresis
Technique that is used to separate DNA fragments (or other macromolecules, such as
RNA or proteins) based on their size and charge.
All DNA molecules have the same amount of charge per mass Separation on size
Gels are made out of agarose and stained with a DNA-binding dye for visualization
3. Gel analysis and DNA sequence determination
Each piece ends with a coloured marker that shows a specific base
By reading the markers from smallest to largest piece, the DNA sequence can be
determined
Sanger sequencing can be used to test for familial variations that are passed from parent to child. Also,
when the focus is on a specific gene that is associated with a known mutation or to detect SNPs.
However, it only works for short regions of the genome.
DNA microarrays are nucleic acid probes that can be labelled and used to detect/visualize DNA (or RNA)
fragments with sequence homology:
1. Analysis of genomic gains and losses by array comparative genomic hybridization (aCGH)
Detects if large regions of the genome have been deleted or duplicated
2. Analysis of DNA variation for mutation detection and single nucleotide polymorphism (SNP
arrays)
3. Analysis of differential gene expression (mRNA -> cDNA)
Advantages of DNA microarrays are that they are reliable, reproducible and can analyse a large number
of genes in one experiment. Disadvantages are that it can’t provide information on position in the
genome or structural information and SNP can’t detect balanced deletions since it can’t determine the
total gene amount, so a diagnosis can be missed.
WES is whole exome sequencing, in which often 0-5 de novo variants are found. NGS is next generation
sequencing, used to determine the order of nucleotides in entire genomes or targeted regions of
DNA/RNA. NGS uses target enrichment, so gene panels focus on a limited number of genes, which is not
the case in WES. The mechanism of NGS is as follows:
1. Library preparation: Adding adaptors
2. DNA library application: Fragments bind to primer
loaded flow cell and bridge PCR reaction amplify
, each bound fragment to produce clusters of fragments. One fluorophore nucleotide is added to
the growing strands.
3. DNA library sequencing: Sequencing of the fluorophores on all fragments
Disadvantages are that it only sequences the exome (2% of DNA), some sequence pieces are too large to
be detected and alignment can be difficult due to repeated areas. Advantages are that it is broad genetic
screening with higher chance of identifying the responsible mutation and it is useful for rare genetic
diseases where the cause may lie in multiple genes.
Variant classification is the process of evaluating the clinical significance of a genetic alteration. There are
5 different classifications:
1. Benign
2. Likely benign
3. Variant of uncertain significance (VUS)
Not enough evidence to determine the classification
4. Likely pathogenic
5. Pathogenic
Benign and likely benign due not contribute to disease. (Likely) pathogenic is are and directly contributes
to disease, so these are clinically relevant. The workflow happens according to ACMG guidelines (=
internationally accepted guidelines for the interpretation of variants):
Identification
Filtering by looking into databases
Prioritising: Likelihood of pathogenicity
Clinical interpretation: Likelihood of causality
o Co-segregation: The transmission to the next generation, of two or more genes in
proximity on the same chromosome
Disadvantages are that is the potential of misclassification and it is hard to diagnose rare disorders due to
lack of data.
Episignatures are patterns of DNA methylation that serve as biomarkers for certain genetic conditions,
especially neurodevelopmental disorders. They are used to classify VUS. The mechanism is:
1. Blood samples
2. DNA isolation
3. DNA methylation profiling: arrays to detect methylation at specific CpG sites across genome
4. Machine learning models
5. Interpretation and diagnosis
Advantages are that it can be used for undiagnosed disorders and disadvantages are that many
episignatures are still unknown and it is a costly process.
RARE DISEASE
HEALTHCARE
All together, an estimated 30 million people are living with a rare disease in 48 countries in Europe. Each
one affects fewer than 1 in 2000 people. There are over 6000 distinct rare disease, of which 72% are
genetic. The other 28% non-genetic, including rare cancers, rare infections and health hazards. Rare
diseases affect 4% of the population in the course of the lives. The onset of 70% of rare disease is in
childhood. Eurordis is a not-for-profit organisation working across borders and diseases to improve the
lives of people living with a rare disease. People living with a rare disease have difficulties with several
activities of daily living. The symptoms can vary and be invisible in many ways. Access to healthcare and
habilitation for people living with a rare disease is complex and hard to manage. People living with a rare
disease have uncovered health and rehabilitation needs.
, European policy
Commission expert group recommendations to support integration of rare disease into social policy:
Member States (MS) should promote measures that facilitate multidisciplinary, holistic,
continuous, person-centred and participative care provision to people living with rare diseases,
supporting them in the full realisation of their fundamental human rights.
Rare disease specificities should be integrated into national systems assessing a person’s level of
functioning, in line with the United Nations Convention on the Right of Persons with Disabilities.
In NL, recognition centers of expertise rare disease (ECZA) started in 2016:
Review by member of scientific advisory board “Zorginstituut Nederland” and patient
organisation
Multidisciplinarity, transition from child-adult, diagnosis, follow-up and treatment
Focus on (multidisciplinary) care paths and research output
5-yearly recognition by VWS (ministry of health)
After recognition of VWS, there can be applied for European Reference Network (ERN) membership.
Goals are collaboration, development of guidelines, strengthen research, and databases, leading to
concentration of expertise and funding.
ERNs harness the collective knowledge and experience of experts, focusing on a common goal to drive
improve access to diagnosis and treatment by providing high-quality healthcare.
EMBRYOLOGY
Neurulation and forebrain patterning
There is a 3 layered embryonic disk. The in-between layer is mesoderm and the top layer is epiblast,
giving rise to ectoderm that becomes part of skin but also develops neural tissue. The primitive streak is a
swamp where cells fall in and go in between the top and bottom layer, forming the mesoderm. The
neural groove forms from the front to the back and forms a tube. The 2 openings at both ends will remain
for a while. Notochord is longitudinal structure in mesoderm that dictates all kinds of
processes that occur. Neural crest tissue will fall apart, disperse throughout the
embryo, resulting in all kinds of cells and it will also form the peripheral nervous
tissue. There is alar plate and basal plate in the neural tube with a groove in
between. The alar plates meet each other in the top plate.
The 3 primary front parts of the neural tube will form the prosencephalon, mesencephalon and
rhombencephalon. There is already an eye primordium (primary eye) and the pituitary is also present.
Genes are expressed in specific parts that are involved in neural patterning to define what is front, back,
head and tail. The 3 primary front parts will develop in 5 brain regions: telencephalon, diencephalon,
mesencephalon, metencephalon and telencephalon.
Hedgehog pathway
Sonic hedgehog (Shh) is de best studied morphogenic ligand of the
hedgehog pathway. The pre-protein is truncated, palmytolized (fatty
tail added) and sterolized before secretion. It is expressed in the
notochord, prechordal plate,
cranial neural crest, limb buds, etc.
It is mainly involved in neural tube
Clinical genetics are needed in the case of rare disorders (< 1:2000). In 80% of the cases, the rare disorder
is monogenetic. There is learned from the rare disease and precision medicine is applied. The basics in
clinical genetics are equal to the basics in personalized medicine. Major subjects in clinical genetics are:
Oncogenetics (hereditary breast or colon cancer) (41%)
Dysmorphology / syndromes (28%)
Prenatal diagnosis / counselling
Ultrasound abnormalities
Familial hereditary disorder
Chromosomal abnormalities
Cardiogenetics (HCM, ARVC, NCCM)
Connective tissue disorders (e.g. osteogenesis imperfecta)
Neurogenetics (M. Huntington, cerebellar ataxias)
Physical examination and diagnosis
When going to the clinical geneticist, a lot of different questions can be asked:
What is the diagnosis / syndrome?
o A person with complaints or the parents
Pregnancy related
o Congenital anomalies syndrome?
o Recurrence risk of familial disorders
o Chromosomal abnormalities (prenatal diagnosis)
Related to own health
o Hereditary disorders that occur later in life (Huntington)
o Increased risk for cancer (e.g. breast cancer, colon cancer)
o Increased risk for sudden cardiac death
Preventive medicine
Targeted therapy
Related to reproduction
o Consanguinity
o Increased risks based on ethnic background (e.g. Tay Sachs disease in Ashkenazi Jewish
people)
o Recurrence risk of familial disorders
Children are referred to a clinical geneticist when they suffer from mental retardation, multiple
congenital abnormalities, dysmorphism, single congenital abnormalities (cleft lip and/or palate), hearing
loss, congenital cataract, or abnormal growth (short stature or obesity).
What does a diagnosis add in children with MR/congenital abnormalities?
Answers on why relieve feelings of guilt
No need for investigations for other causes
Information on prognosis, treatment
Qualify for support, school options etc
Parental contact
Family members
Recurrence risk, reproductive choices
o Deciding not to have (more) children
o Accepting the risk
o Adoption
o Donor sperm or eggs
o Prenatal diagnosis (NIPT, CVS, AP), followed (or not) by termination of pregnancy
, o Pre-implementation genetic testing (PGT) Only use unaffected embryos
o Adapting partner choice (culturally related)
Clinical genetics is a broad specialism. Family history and physical examination are important for
diagnosis. Counselling before pregnancy is recommended. Clinical genetics provide information on which
counselees can base their own decisions. A genetic diagnosis may enable targeted therapy.
Diagnostic testing
PCR is the exponential amplification of a specific target piece of DNA.
There is a three step synthesis reaction:
1. Desaturation of template to single strands
2. Annealing of primers to each original strand (complementary
binding of the primers to the template)
3. Extension of the new DNA strands from the primers onwards
Needed for PCR are Taq polymerase, primers (forward and reverse),
template DNA, and nucleotides (dNTPs).
In Sanger sequencing, DNA sequencing is used to determine the precise order of the nucleotides of a
given DNA fragment. The reaction mixture consists of primer, DNA-template, DNA pol, dNTPs and ddNTPs
with fluorochromes. The mechanism works as follows:
1. DNA-chain termination via PCR
DNA is copied using special building blocks
Some stop the DNA from growing further
Creates many DNA pieces of different lengths
2. Size separation by gel electrophoresis
Technique that is used to separate DNA fragments (or other macromolecules, such as
RNA or proteins) based on their size and charge.
All DNA molecules have the same amount of charge per mass Separation on size
Gels are made out of agarose and stained with a DNA-binding dye for visualization
3. Gel analysis and DNA sequence determination
Each piece ends with a coloured marker that shows a specific base
By reading the markers from smallest to largest piece, the DNA sequence can be
determined
Sanger sequencing can be used to test for familial variations that are passed from parent to child. Also,
when the focus is on a specific gene that is associated with a known mutation or to detect SNPs.
However, it only works for short regions of the genome.
DNA microarrays are nucleic acid probes that can be labelled and used to detect/visualize DNA (or RNA)
fragments with sequence homology:
1. Analysis of genomic gains and losses by array comparative genomic hybridization (aCGH)
Detects if large regions of the genome have been deleted or duplicated
2. Analysis of DNA variation for mutation detection and single nucleotide polymorphism (SNP
arrays)
3. Analysis of differential gene expression (mRNA -> cDNA)
Advantages of DNA microarrays are that they are reliable, reproducible and can analyse a large number
of genes in one experiment. Disadvantages are that it can’t provide information on position in the
genome or structural information and SNP can’t detect balanced deletions since it can’t determine the
total gene amount, so a diagnosis can be missed.
WES is whole exome sequencing, in which often 0-5 de novo variants are found. NGS is next generation
sequencing, used to determine the order of nucleotides in entire genomes or targeted regions of
DNA/RNA. NGS uses target enrichment, so gene panels focus on a limited number of genes, which is not
the case in WES. The mechanism of NGS is as follows:
1. Library preparation: Adding adaptors
2. DNA library application: Fragments bind to primer
loaded flow cell and bridge PCR reaction amplify
, each bound fragment to produce clusters of fragments. One fluorophore nucleotide is added to
the growing strands.
3. DNA library sequencing: Sequencing of the fluorophores on all fragments
Disadvantages are that it only sequences the exome (2% of DNA), some sequence pieces are too large to
be detected and alignment can be difficult due to repeated areas. Advantages are that it is broad genetic
screening with higher chance of identifying the responsible mutation and it is useful for rare genetic
diseases where the cause may lie in multiple genes.
Variant classification is the process of evaluating the clinical significance of a genetic alteration. There are
5 different classifications:
1. Benign
2. Likely benign
3. Variant of uncertain significance (VUS)
Not enough evidence to determine the classification
4. Likely pathogenic
5. Pathogenic
Benign and likely benign due not contribute to disease. (Likely) pathogenic is are and directly contributes
to disease, so these are clinically relevant. The workflow happens according to ACMG guidelines (=
internationally accepted guidelines for the interpretation of variants):
Identification
Filtering by looking into databases
Prioritising: Likelihood of pathogenicity
Clinical interpretation: Likelihood of causality
o Co-segregation: The transmission to the next generation, of two or more genes in
proximity on the same chromosome
Disadvantages are that is the potential of misclassification and it is hard to diagnose rare disorders due to
lack of data.
Episignatures are patterns of DNA methylation that serve as biomarkers for certain genetic conditions,
especially neurodevelopmental disorders. They are used to classify VUS. The mechanism is:
1. Blood samples
2. DNA isolation
3. DNA methylation profiling: arrays to detect methylation at specific CpG sites across genome
4. Machine learning models
5. Interpretation and diagnosis
Advantages are that it can be used for undiagnosed disorders and disadvantages are that many
episignatures are still unknown and it is a costly process.
RARE DISEASE
HEALTHCARE
All together, an estimated 30 million people are living with a rare disease in 48 countries in Europe. Each
one affects fewer than 1 in 2000 people. There are over 6000 distinct rare disease, of which 72% are
genetic. The other 28% non-genetic, including rare cancers, rare infections and health hazards. Rare
diseases affect 4% of the population in the course of the lives. The onset of 70% of rare disease is in
childhood. Eurordis is a not-for-profit organisation working across borders and diseases to improve the
lives of people living with a rare disease. People living with a rare disease have difficulties with several
activities of daily living. The symptoms can vary and be invisible in many ways. Access to healthcare and
habilitation for people living with a rare disease is complex and hard to manage. People living with a rare
disease have uncovered health and rehabilitation needs.
, European policy
Commission expert group recommendations to support integration of rare disease into social policy:
Member States (MS) should promote measures that facilitate multidisciplinary, holistic,
continuous, person-centred and participative care provision to people living with rare diseases,
supporting them in the full realisation of their fundamental human rights.
Rare disease specificities should be integrated into national systems assessing a person’s level of
functioning, in line with the United Nations Convention on the Right of Persons with Disabilities.
In NL, recognition centers of expertise rare disease (ECZA) started in 2016:
Review by member of scientific advisory board “Zorginstituut Nederland” and patient
organisation
Multidisciplinarity, transition from child-adult, diagnosis, follow-up and treatment
Focus on (multidisciplinary) care paths and research output
5-yearly recognition by VWS (ministry of health)
After recognition of VWS, there can be applied for European Reference Network (ERN) membership.
Goals are collaboration, development of guidelines, strengthen research, and databases, leading to
concentration of expertise and funding.
ERNs harness the collective knowledge and experience of experts, focusing on a common goal to drive
improve access to diagnosis and treatment by providing high-quality healthcare.
EMBRYOLOGY
Neurulation and forebrain patterning
There is a 3 layered embryonic disk. The in-between layer is mesoderm and the top layer is epiblast,
giving rise to ectoderm that becomes part of skin but also develops neural tissue. The primitive streak is a
swamp where cells fall in and go in between the top and bottom layer, forming the mesoderm. The
neural groove forms from the front to the back and forms a tube. The 2 openings at both ends will remain
for a while. Notochord is longitudinal structure in mesoderm that dictates all kinds of
processes that occur. Neural crest tissue will fall apart, disperse throughout the
embryo, resulting in all kinds of cells and it will also form the peripheral nervous
tissue. There is alar plate and basal plate in the neural tube with a groove in
between. The alar plates meet each other in the top plate.
The 3 primary front parts of the neural tube will form the prosencephalon, mesencephalon and
rhombencephalon. There is already an eye primordium (primary eye) and the pituitary is also present.
Genes are expressed in specific parts that are involved in neural patterning to define what is front, back,
head and tail. The 3 primary front parts will develop in 5 brain regions: telencephalon, diencephalon,
mesencephalon, metencephalon and telencephalon.
Hedgehog pathway
Sonic hedgehog (Shh) is de best studied morphogenic ligand of the
hedgehog pathway. The pre-protein is truncated, palmytolized (fatty
tail added) and sterolized before secretion. It is expressed in the
notochord, prechordal plate,
cranial neural crest, limb buds, etc.
It is mainly involved in neural tube
