11 Arrays, NGS, Epigenetics

• a method to screen specimens for variation
• include comparative genomic arrays (SNPs), gene expression arrays (micro), & NGS (Next Generation Sequencing - high throughput sequencing)

• the first test of choice when there isn’t a clear clinical picture
• when there’s developmental delay
• when dysmorphic features are present but don’t fit any syndrome
• to characterize tumors
• to link genotype & phenotype (via GWAS - genome-wide association studies)

• when all other testing is normal
• takes longer, is slower, & more costly

• identifies differences between control DNA & patient (test) DNA
• can pick up deletions & duplications (has a limited application - also picks up deletions better than duplications)
• identified changes MAY be related to the reasons for testing, or may just be a polymorphism

• they may NOT be able to detect low level mosaicism
• they can’t detect balanced translocations
• when you’ve extracted DNA from a cell & using a CGH array, all you’re asking is ‘how much DNA do I have?’
• it can’t distinguish between someone who has 2 perfectly normal intact chromosomes, & someone with a BALANCED translocation

• many regions between individuals are identical
• however there’s as much as 12% variability between individuals
• variability was either polymorphisms (changes in sequence with no phenotypic effect) or copy number variation (more or fewer copies of a specific DNA region)

• 2.4 million
• this accounts for less than 1% of our genome
• occur because of the wobble in the genetic code

• each may or may not result in a change in the amino acid sequence
• each chromosome will have MANY SNPs
• these changes are inherited like mendelian characteristics (could be added or removed by mutations or deletions)
• they can help identify a person’s origin because of similarities within a population (founder effect, isolation)
• can be useful for gene mapping - are variable, have great resolution

when mutations had by early ancestors persist in a population (eg. Ashkenazis)

• if a group of individuals are isolated either by choice or by geography, there’s a limited number of individuals breeding in that group
• therefore they’re going to become more homogeneous over time than if there was outbreeding

• single nucleotide polymorphisms observed in groups
• eg. SNPs 1, 2, 3, 4, 5, 6, 7 - if you look for 3, you’ll ALWAYS fine 1, 2, 4, 5, 6, 7

• asks which SNPs are present
• assumes that all SNPs occur with similar frequency
• expect heterozygosity, so tells you if there’s LOH (loss of heterozygosity)
• expect to have 2 copies at EACH position, one from the mother & one from the father
• it’s easier to see deletions than duplications

SNP arrays provide more information because they report the amount of DNA

• SNP Arrays will only identify ISOdisomies (result of meiosis II error)
• heterodisomy (meiosis I error) can’t be recognized

• widely used to characterize TUMORS
• can have gain of a DNA region that doesn’t result in overprotection of protein, so it’s better to look at the END result (DNA expression into proteins) than to just look at the DNA

• can either sequence the entire genome (WGS) or the region of the genome that just encodes the genes (exome = WES)
• compare results to control databases
• if NGS finds a change not present in a control sample, it’s probably related to the patient’s presentation

• a mutation (AD) or 2 mutations (AR) in a gene related to the phenotype
• variant(s) of unknown significance (VUS) - mutations POSSIBLY related to the phenotype
• mutations/VUSs unrelated to the phenotype
• medically actionable mutations
• carrier status for Mendelian disorders
• deleterious mutations in genes with no disease association

an “actionable” gene in adults is one that could potentially cause harm, & surveillance/prevention was shown to improve outcomes

it’s much more difficult to know what’s going on with quantitative traits like Heart disease or diabetes versus single gene diseases like CF or Achondroplasia

• 85%
• there are ~180,000 exons (account for 3% of the genome, aka 22,000 genes)
• that leaves ~15% occurring elsewhere

• Kabuki Syndrome
• short stature, microcephaly, developmental delay, hearing loss, heart/kidney defects, feeding difficulties, characteristic facial features (long palpebral fissures, lateral eversion of eyelids, sparse & arched eyebrows, large ears), characteristic fat deposition on finger tips

• optimally send parent samples along with patient samples to interpret variants
• if healthy parents carry a variant, it’s unlikely that it’s related to the patient’s phenotype

finds mutations definitely related to the phenotype, possibly related to the phenotype (VUSs), medically actionable mutations, & carrier status for Mendelian disorders

• will find mutations in genes unrelated to phenotype
• VUS unrelated to the given phenotype
• deleterious mutations in genes that have nothing to do with the disease in question

• modification of gene expression without changing the DNA sequence
• may explain why there’s VARIABLE EXPRESSIVITY in autosomal dominant disorders

• 1. Methylation: silences gene expression
• 2. Histone Modification: can enhance or suppress gene expression
• 3. Nucleosome Positioning: can “tighten” so coding/transcription areas are inaccessible
• 4. ncRNA (non-coding): inhibits mRNA

• loss upstream of a transcription start site → gene activation
• loss downstream of a transcription start site → blocks the gene itself, therefore causes gene repression
• upstream: opposite the direction of transcription
• downstream: in the direction of transcription

lack of concordance between monozygotic twins