NGS
- Gene%c disorders are frequent
Iden%fying gene%c cause in know gene%c disorders
Miller syndrome
- Limb abnormali%es
- Eye lid problems
- …
- Different pa%ents reported independently - > gene%c disorder
- What is the cause of the gene%c disorder?
o Parents are related -> autosomal recessive
How to proceed?
- Look at a few families
o Look at the variants in all the desease pa%ents
o There is one gene that is responsible or the variant is specific for that pa%ent?
§ Do you need to sequence everything?
• No, a small part are coding sequencing -> whole exome sequencing (1-
2% of the genome is the exome)
Whole exome sequencing
- All genomic sequences
o Blue = exons
o Red = non coding sequences
- Shearing DNA -> adap%ng liga%on -> add probes and these contain all the exonic
sequences -> bio%n label -> you capture -> you mix the two sequences -> bind to the
coding sequences -> take out the bio%n, so all the coding sequences
- Shearing -> add adapters -> probes bound to the array (only probes for the exonic
sequences) -> blue ones are bound -> red ones are washed way -> whole genome
sequencing
- S%ll expensive
, Databases for normal varia<on (control popula<ons sequences0
o Substract the normal varia%on -> you see abnormali%es
Muta<ons in DHODH gene
Iden%fying novel, as yet unknown gene%c disorders
Trio approach
- De novo muta%on = muta%on in the child, but not in the parents
o Like down syndrome, but also intellectual disorders
- Look at the father, mother and child
o Abnormality also present in the mother -> not the cause for the disease
§ Same for father
o Abnormality in child, but not in parents -> related to the disease
- Not all the novel varia%ons cause the disease
Variant calling
- Sequence father, mother and child
- Lot of raw data -> filtering steps with bioinforma%cs-> 1 of 2 de novo variants
Example from own research
- Sequence all the exomes, from the child, the father and mother
- 4 bp del in ADNP was found
o Causes frameshiV introducing stop codon
o Looked in control databases -> found nothing
ADNP gene
- Expressed in brain
- Zinc fingers/homeobox domain: poten%al transcrip%on factor
- Involved in neurogenesis
- Involved in heart development
- Homozygous KO mice are embryonically lethal
- Heterozygous KO mice have cogni%ve & behavioral problems
Look to other muta<ons
- One descrip%on of a group that found two muta%ons in the gene
o MIP sequencing to sequence large numbers of candidate genes
- 10 pa%ents with trunca%ng muta%ons in ADNP
- The de novo muta%ons in ADNP cause new au%sm syndrome
How about genes with only a single muta<on
- MIPs technology comes in the picture
Would you not like to screen a large set of pa%ents ?
- MIPs is useful for this
- Gene%c disorders are frequent
Iden%fying gene%c cause in know gene%c disorders
Miller syndrome
- Limb abnormali%es
- Eye lid problems
- …
- Different pa%ents reported independently - > gene%c disorder
- What is the cause of the gene%c disorder?
o Parents are related -> autosomal recessive
How to proceed?
- Look at a few families
o Look at the variants in all the desease pa%ents
o There is one gene that is responsible or the variant is specific for that pa%ent?
§ Do you need to sequence everything?
• No, a small part are coding sequencing -> whole exome sequencing (1-
2% of the genome is the exome)
Whole exome sequencing
- All genomic sequences
o Blue = exons
o Red = non coding sequences
- Shearing DNA -> adap%ng liga%on -> add probes and these contain all the exonic
sequences -> bio%n label -> you capture -> you mix the two sequences -> bind to the
coding sequences -> take out the bio%n, so all the coding sequences
- Shearing -> add adapters -> probes bound to the array (only probes for the exonic
sequences) -> blue ones are bound -> red ones are washed way -> whole genome
sequencing
- S%ll expensive
, Databases for normal varia<on (control popula<ons sequences0
o Substract the normal varia%on -> you see abnormali%es
Muta<ons in DHODH gene
Iden%fying novel, as yet unknown gene%c disorders
Trio approach
- De novo muta%on = muta%on in the child, but not in the parents
o Like down syndrome, but also intellectual disorders
- Look at the father, mother and child
o Abnormality also present in the mother -> not the cause for the disease
§ Same for father
o Abnormality in child, but not in parents -> related to the disease
- Not all the novel varia%ons cause the disease
Variant calling
- Sequence father, mother and child
- Lot of raw data -> filtering steps with bioinforma%cs-> 1 of 2 de novo variants
Example from own research
- Sequence all the exomes, from the child, the father and mother
- 4 bp del in ADNP was found
o Causes frameshiV introducing stop codon
o Looked in control databases -> found nothing
ADNP gene
- Expressed in brain
- Zinc fingers/homeobox domain: poten%al transcrip%on factor
- Involved in neurogenesis
- Involved in heart development
- Homozygous KO mice are embryonically lethal
- Heterozygous KO mice have cogni%ve & behavioral problems
Look to other muta<ons
- One descrip%on of a group that found two muta%ons in the gene
o MIP sequencing to sequence large numbers of candidate genes
- 10 pa%ents with trunca%ng muta%ons in ADNP
- The de novo muta%ons in ADNP cause new au%sm syndrome
How about genes with only a single muta<on
- MIPs technology comes in the picture
Would you not like to screen a large set of pa%ents ?
- MIPs is useful for this
