DD genomics

Question 1

DD are individually rare but collectively common
A. True
B. False

Answer 1

A. True

Question 2

why is molecular/genetic diagnosis is important DD?*

Answer 2

1. management - informs management (sometimes)
2. treatment - helps to access special needs services
3. prognosis - informs prognosis (sometimes)
4. reproductive counselling - informs future reproductive decisions

Question 3

why majority of DD remain undiagnosed after the standard NHS testing?

Answer 3

1. diverse clinical features
2. diverse molecular causes and inheritance
3. majority is genetic

Question 4

what are the range of phenotype associated with DD?*

Answer 4

1. intellucrtual disability (87%)
2. seizures (24%)
3. autism spectrum disorder (10%)
4. congenital heart defects (11%)
5. hearing impairment (7%)
6. oral cleft (6%)
7. scoliosis (5%)
8. polydactyl (1%)

Question 5

what are the range of genes and genetic variants that can cause DD*

Answer 5

• > 1500 genes linked to DD
• genetic variants includes
  1. single nucleotide variant (SNV)
  2. insertion/deletion (indels)
  3. copy number variations (CNVs)
  4. chromosomal aneuploidies

Question 6

what are three main types of DNA variants based on location?

Answer 6

1. intronic/intergenic (non-coding)
   - between genes or axons
   - do not affect protein directly
   - could affect regulation of transcription or splicing
2. gene/coding/exonic variants
   - silent variant (synonymous)
   - missense variant (non-synomous) changes AA
   - nonsense variant resulting in premature STOP
   - frameshift variants altering reading frame (deletions and insertions)
3. multi-expo/multigene
   - structural variant can affect multiple genes e.g. copy number

Question 7

what is two standard diagnostic testing for DD?*

Answer 7

1. molecular genetics - sequencing of genes for genotyping specific small mutations
   - uses sequencing and PCR
   - detects SNVs and indels
   - requires a priori hypothesis
2. cytogenetics - evaluating the genome for large structural changes that could reflect/explain the phenotype
   - uses karyotyping, FISH, microarrays
   - detects CNVs, chromosomal anueploidies, translocations, inversions
   - do not require a priori hypothesis of cause

Question 8

name two DD

Answer 8

1. 16p11.2 mcirodeletion/duplication (de novo dominant mutation)
2. Barnet-biedl syndrome (BBS) (autosomal recessive inheritance)
3. Prader willi syndrome
4. down syndrome
5. SZ

Question 9

what are the features of 16p11.2 micro deletion/duplication?

Answer 9

1. CNVs (deletion/duplication-indels)
2. de novo dominant mutations
3. 7-8MB of DNA, >60 genes
4. ID, autism, developmental delay

Question 10

what does duplication and deletion in 16p11.2 micro deletion/duplication result in?

Answer 10

duplication -> risk of being underweight

deletion - risk of obesity

Question 11

what are the features of BBS?

Answer 11

1. SNVs and indels in one of ~20 genes

2. autosomal recessive inheritance

Question 12

what are the modern diagnostic testing in DD?

Answer 12

1. standard diagnostic testing
2. targeted next generation sequencing
3. genome wide sequencing using NGS

Question 13

what is trio sequencing approach? and why is it powerful?*

Answer 13

genetic data from child and both parents are collected which hugely reduces number of candidate casual variants

Question 14

how is exome sequencing used to diagnose rare diseases?*

Answer 14

• rare mutations in ~4000 genes are known to cause disease and there are many genes with unknown function
• therefore, exome sequencing enables the discovery of novel disease causing variation in previously unclassified genes

Question 15

what are the challenges in working with GWS data?*

Answer 15

1. enormous amount of genetic variation
2. every genome contains a novel variation
3. most genetic variation is benign
4. phenotypic and genotypic heterogeneity
5. disease inheritance and mechanism is unknown
6. phenotypes may have more than one genetic cause

Question 16

what is DDD?

Answer 16

deciphering developmental disorder is UK-collaborative studies of families, NHS and Sanger institute

Question 17

what is Coffin-Siris (ARID1B) syndrome

a. caused by
b. prevalence
c. phenotypic spectrum
d. treatments

Answer 17

1. cause by de novo loss of function of ARID1B - chromatin remodeller; changes DNA accessibility for gene expression
2. top gene in DDD - diagnosis in 0.7% cases
3. broad phenotypic spectrum - developmental delay, speech delay, abnormalities of fifth (pinky) fingers or toes, short sighted, feeding difficulties; overlapping with other disorders
4. no current treatments - can counsel parents about low recurrence risk

Question 18

what is Coffin-Siris (ARID1B) syndrome

1. caused by
2. its prevalence and rate of diagnosis
3. phenotypic spectrum
4. treatments

Answer 18

1. cause by de novo loss of function of ARID1B - chromatin remodeller
2. top gene in DDD - diagnosis in 0.7% cases
3. broad phenotypic spectrum - developmental delay, speech delay, abnormalities of fifth (pinky) fingers or toes, short sighted, feeding difficulties; overlapping with other disorders
4. no current treatments - can counsel parents low recurrence risk

Question 19

how to find out more DD genes?

Answer 19

1. recruit more patients
2. persuade other researchers to give us their data (i.e. collaborate with other similar cohorts)
3. share our data with other researchers

Question 20

what is the cause and diagnosis for KMT2B mutations?

Answer 20

1. de novo mutations cause dystonia and ID
2. diagnosis involve es DNA packing and gene expression
3. patients successfully treated with deep brain stimulation

Question 21

what is the advantage of NGS and how is it beneficial for us?*

Answer 21

1. has enabled simultaneous sequencing of all genes
2. many new disease caused by genes are discovered
3. large burden of casual de novo mutations through trio sequencing
4. helps target treatments

Question 22

what are the ethical issues in sequencing and what is it caused by?

Answer 22

• caused by data size and broad applicability
• since variation interpretation is challenging so need to share data
• concerns for handling incidental and secondary findings

Question 23

How many variants are in an average genome?
A. 3-4 billion
B.4-5 million
C.500,000
D.20,000

Answer 23

B. 4-5 million

Question 24

Which of the following variants change the number of basepairs in the genome?

1. SNVs
2. Indels
3. CNVs
4. Translocations
5. Inversions
6. Aneuploidies

Answer 24

indels, CNVs, aneuplodies

Question 25

Why are rare variants rare? 1. They are new OR very damaging 2. They are new AND very damaging 3. They are old OR not very damaging 4. They are old AND not very damaging

Answer 25

they are new OR very damaging

Question 26

what are the endogenous and exogenous sources of variation?

Answer 26

``` A. endogenous - 1. hydrolysis 2. deamination of methylated cytosines 3. recombination error 4. segregation error 5. base pair mismatch during replication 6. replication slippage B. exogenous 1. chemical agents 2. UV radiation ```

Question 27

what is the impact of increasing paternal and maternal age on developmental syndromes?

Answer 27

1. there is increased risk of sequence mutations with increase in paternal (mainly) and maternal age, mostly due to replication errors in mitosis in developmental syndromes (e.g. dominant single gene disorders) 2. structural mutations increase with increase in maternal age due to NDJ or recombination errors in meiosis I and is seen in trisomies (downs syndrome)

Question 28

what is genotyping and what are its types?*

Answer 28

genotyping is determining which genetic variants are present in an individual genome 1. arrays: only assays specific type of variants a. SNP-chip - type of DNA microarray that detect polymorphisms in a given population b. array-CGH (comparative genomic hybridisation) - determines CNVs of specific regions on microarray 2. sequencers: only assays variants within specific sequenced region of DNA

Question 29

what is sequencing and what are its types?*

Answer 29

it is determining the order of basepairs in a region of DNA 1. Sanger sequencing - targets specific exons or a single gene 2. NGS - targets multiple genes or whole genome

Question 30

why is NGS effective for diagnosing paediatric rare diseases?*

Answer 30

1. lots of different genes cause overlapping of phenotype 2. reduced reproductive fitness therefore, expect ultra-rare variants - high burden of de novo mutations (which are easy to find) - recessive disease enriched in consanguineous (blood related) populations - casual variants (variants and types) will not be present in adults 3. phenotype arises early, less likely to be influenced by environmental factors

Question 31

what type mutations are significant in in SZ/DD?

Answer 31

high burden of de novo mutations

Question 32

ASD is a monogenic disease? A. True B. False

Answer 32

True, it is monogenic and syndromic forms

Question 33

what are features of ASD?

Answer 33

1. monogenic and syndromic forms 2. complex multifactorial causes 3. high burden of de novo mutations and large CNVs

Question 34

what are the factors influencing the onset of ASD?

Answer 34

1. familial background 2. high risk events - non-genetic factors like paternal age, de Nono mutations and recessive mutations 3. varying clinical manifestations - comorbidities and overlaping of diseases

Question 35

what is the prevalence and heritability rate of ASD?

Answer 35

prevalence ~1.5% | heritability ~50-59%

Question 36

what is the most common phenotype in DD?

Answer 36

intelluctual disability

Question 37

what value is considered as intellectual disability?

Answer 37

IQ<70

Question 38

why is family trio exam sequencing useful for diagnosing DD's?

Answer 38

1. determining if recessive variants in child are inherited from carrier parents 2. finding de novo variants 3. finding new disease genes 4. exclusion of benign inherited variants

Question 39

what is the common cause of 1. dominant DD and 2. recessive DD?

Answer 39

1. dominant DD - de novo heterozygous variants | 2. recessive DD - inherited variants

Question 40

how do de novo SNVs usually cause disease?

Answer 40

through loss of function in dominant disease genes

Question 41

what is haploinsufficiency?

Answer 41

only having a single copy of the gene (halpoid) is not enough for normal function so the loss of function mutations cause a dominant phenotype

Question 42

what information can be used to determine if a variant is pathogenic or not?*

Answer 42

1. comparing other patients with the same variant in the same gene 2. use statistical methods to show enrichment of variants in disease cohort 3. evaluating whether variant is present in normal controls 4. evaluating in vitro and in vivo functional data to show the effect of variant 5. testing segregation in family members and other affected families

Question 43

what is the risk of recurrence of in the following conditions for disease in future pregnancies? 1. de novo dominant 2. inherited autosomal dominant 3. autosomal recessive 4. X-linked recessive

Answer 43

1. de novo dominant - <1% 2. inherited autosomal dominant - 50% 3. autosomal recessive - 25% 4. X-linked recessive - 50%

Question 44

what is NGLY1 and what does it deficiency result in?

Answer 44

- it is an autosomal recessive gene called N-glycanase 1 - its deficiency results in impaired ability to deglycosylate proteins, cells accumulate misfiled glycoproteins - its results in physical nd neurological symptoms like lack of tears, developmental delay, movement disorder and elevated liver enzymes

Question 45

what are the important issues that should be discussed with the family when taking consent for trio sequencing?*

Answer 45

- consent should be voluntary and informed - the issues for discussing during consent are 1. capacity for consent/assent 2. likelihood of finding (and not finding a diagnosis) 3. timescale for finding a diagnosis 4. potential for treatment 5. implications for other families 6. importance of sharing data 7. potential for incidental findings

Question 46

why only specific genes are highlighted in a condition and how are other variants filtered/excluded?

Answer 46

- variants are excluded if 1. low quality 2. common 3. silent/non-coding 4. not in known DD disease causing gene 5. do not fit with family history

Question 47

what does the candidate variant usually include?

Answer 47

1. de novo mutations (in dominant genes) | 2. homozygous variants (in recessive genes)

Question 48

why one mutation is determined to be in the diagnosis and not another one?

Answer 48

1. a better fit between the phenotype and the associated gene of the patient (KMT2B) - a better fir between the mechanism of disease and type of mutation (KMT2B) in the gene - similarity between the patients phenotype and other patient with that mutation - definitely pathogenic

Question 49

why is ARID1B not considered for the diagnosis of Billy's case?

Answer 49

- Missense mutations in ARID1B usually don’t cause disease, only LOF mutations - Unstructured protein that is tolerant to missense changes - No reports of dystonia in literature, no fiFh finger abnormality in Billy May explain part of phenotype?? - Likely benign variant that does not contribute to the phenotype

Question 50

what is decipher?

Answer 50

1. DECIPHER is a web-based platform which helps clinical and research teams to assess pathogenicity and to share patient data 2. Integrates genetic and phenotypic data from population and patients 3. Thousands of variants, mostly in patients with DD

Question 51

SNVs within genes can result in: 1. Altered amino acid 2. Deletion of an exon 3. Truncated protein 4. No effect (silent) 5. Frameshi@ mutation 6. Duplication of an exon 7. Trinucleotide repeat expansion

Answer 51

1. altered AA 3. truncated protein 4. no effect (silent)

Question 52

what is non-disjunction?

Answer 52

Failure of homologous chromosomes or sister chromatids to separate equally into daughter cells during mitosis/meiosis

Question 53

what are the features of third generation DNA sequencing?

Answer 53

1. long single module reads (>10,000 bp) - can sequence large repeated regions and detect trinucleotide expansion 2. can detect epigenetic markers (DNA methylation) 3. higher per base error rate than NGS - not yet accurate/cheap enough for WES/WGS

Question 54

what is exome sequencing?

Answer 54

sequencing all the axons of all the genes in a genome

Question 55

which NGS does exome sequencing use and bases can it read per person?

Answer 55

1. uses second generation NGS | 2. 10 million reads of ~75-150 bases per person

Question 56

Why is NGS so powerful for diagnosing rare disease?*

Answer 56

1. Most rare diseases have a genetic component 2. Rare diseases are caused by many different genes 3. Rare diseases are caused by many different types of mutations 4. NGS offers a gene-agnostic approach to testing

Question 57

what are the features of NGS?

Answer 57

1. massive parallel sequencing of short fragments ("read") of DNA 2. fast, cheap, high throughput but computationally intensive 3. can sequence whole genome and detect most (but not all) variants