Genetic Testing 101: Interpreting the Chromosomes Kristin Lindstrom, MD Division of Genetics and Metabolism Phoenix Children s Hospital AzAAP Pediatrics in the Red Rocks
I have no disclosures for this presentation. I do not plan to discuss unapproved or off label use of products
Overview 1. Chromosome Analysis 2. FISH 3. Chromosome Microarray For each, we will review: What they are and are not useful for When to send them in a primary care setting How to begin discussion and work up with family We are focusing on chromosome-based testing because: Chromosome disorders are extremely common It is the most common genetic testing that would likely be done in the primary care setting Even in a genetics clinic visit chromosome testing is usually part of our first line work-up unless a very specific genetic disorder is suspected
Quick Caveat The ability to send genetic testing from the primary care office, and what lab(s) you send to may be very dependent on the patient s specific insurance, and what that plan covers (which seems to be a moving target) If you re not sure if a certain test is covered under a patient s insurance, it is prudent to figure this out before sending the test, as a common complaint from families is that they are hesitant to do further genetic testing because they are fighting a huge bill from the testing done in the PCP s office
My Favorite Analogy Chromosome Gene
Relative Sizes in basepairs Haploid human genome (one egg/sperm)? 3 gigabases (billion basepairs) Largest chromosome? Chromosome 1: 249 megabases (million basepairs) Smallest chromosome? Chromosome 21 (doh!): 48 megabases One band on a chromosome analysis? 5-10 megabases Largest gene? Dystrophin: 2 megabases
Chromosome Disorders More common than single gene disorders 1% of live births 2% of pregnancies with prenatal dx in women >35 10% of stillbirths and of neonatal deaths 50% of spontaneous 1 st trimester abortions Common cause of miscarriage, congenital malformations and intellectual disability
Chromosome Analysis Birds eye view, done in metaphase p for petite/pequena (short arm) q because q comes after p 1 band = 5-10 Megabases (million base pairs) Numbering system has become more complicated over time as the band levels have improved
Chromosome Analysis (Karyotype) What is it good for? Trisomy/Monosomy Relatively large rearrangements Balanced translocations Mosaicism need to specify, so more cells are counted (typically, 20 counted) What does it not tell you? Specific breakpoints Microdeletion syndromes Anything about genes
Balanced Translocation
Counseling Scenario You are seeing a couple for a preconception visit because one of them found out they were a balanced translocation carrier. What do you quote them as their risk of having a child born with an unbalanced translocation (= syndrome)? 10-15% for a female carrier 5% for a male carrier Affected sperm less fit??
Robertsonian Translocations
Robertsonian Translocations Which chromosomes are acrocentric? 13, 14, 15, 21, 22 What are the phenotypes of Robertsonian Translocations? Trisomy 13 and 21 Miscarriage What is the risk of having a child born with Down Syndrome if a parent has a 21/21 Robertsonian Translocation? 100%!
Chromosome Analysis: When to Send It If you suspect Down Syndrome This is the only test you need to send Send even when an amniocentesis or NIPT was done prenatally If you suspect Turner Syndrome Ruling out mosaicism is important, as there are many possible types Send even when an amniocentesis or NIPT was done prenatally A newborn with multiple congenital anomalies Looking for an unbalanced translocation or large pieces of chromosome extra or missing (including trisomy 13 and 18) May want to send at the same time as a CMA A parent with multiple miscarriages Looking for a balanced translocation Other conditions that can be diagnosed on chromosome analysis: Cri-du-chat, Wolf-Hirschhorn, Trisomy X, Klinefelter Syndrome, 47,XYY
You get an abnormal chromosome analysis: what next (other than genetics referral)? Down Syndrome: Health Supervision for Children With Down Syndrome. Pediatrics 2011;128;393 Echocardiogram CBC, TSH Hearing screen, ENT referral, Ophthalmology DS specific growth charts Turner Syndrome: Clinical practice guidelines for the care of girls and women with Turner syndrome: proceedings from the 2016 Cincinnati International Turner Syndrome Meeting. European Journal of Endocrinology (2017) 177, 1 70 Echocardiogram, cardiology referral Endocrinology referral Renal ultrasound TS specific growth charts
You get an abnormal chromosome analysis: what next (other than genetics referral)? Unbalanced translocation (1 piece of chromosome missing and 1 piece of chromosome extra) or a large single piece of chromosome missing or extra (including trisomy 13 & 18): Echocardiogram/cardiology evaluation Renal ultrasound Feeding evaluation Hearing and vision evaluations Assess for possible seizures Developmental pediatrics evaluation
Fluorescent In Situ Hybridization (FISH) Specially designed fluorescent probes that hybridize to metaphase or interphase chromosomes Confirm if specific pieces identified on karyotype/array are really extra/missing Confirm where duplications insert
FISH: Limited Utility in Primary Care I personally only use FISH to look for the SRY gene (in the setting of a virilized girl with Turner syndrome or other DSD), or for confirmation of CMA abnormalities in parents I almost never use FISH as a diagnostic tool It s not the best test for Down Syndrome or Turner syndrome because you still need the chromosome analysis to tell you about translocations and mosaicism A CMA is better for DiGeorge/22q11.2 deletion syndrome and Williams Syndrome because you get additional important information
Chromosome Microarray (CMA) Answers the question: Are there any (big or small) chromosome pieces extra or missing? What they can find: Microdeletion/duplication syndromes (22q11.2, Williams Syndrome) Small, random deletions/duplications Whole gene deletions (sometimes) Uniparental disomy and loss of heterozygosity The cannot detect: Mutations or tiny deletions/duplications within a gene Trinucleotide repeats (like Fragile X) Structural anomalies (translocations, inversions, ring chromosomes) They come with the problem of: Variants of unknown significance (VUS). These are common! Size? Deletion or duplication? Genes involved? Inheritance?
CMA: CGH vs. SNP Comparative Genomic Hybridization Single Nucleotide Polymorphism 3,000,000 SNPs A or B At any given SNP, your genotype can be: AA AB/BA BB
SNP CMA Relative amount of DNA BB AB/BA AA
SNP CMA: Copy Number Variant (CNV) duplication BBB BBA AAB AAA
SNP CMA: Loss of Heterozygosity 1. Consanguinity Relative amount of DNA BB AB/BA AA 2. Uniparental Disomy Relative amount of DNA BB AB/BA AA
ACMG Guidelines for CMA CMA testing is recommended as a first-line test in the initial postnatal evaluation of individuals with the following: A. Multiple anomalies not specific to a well-delineated genetic syndrome. B. Nonsyndromic Developmental Delay/Intellectual Disability C. Autism spectrum disorders. About 20-25% of these children will have a pathogenic CMA finding
CMA: When to send it (and not send it) Send: If you suspect a specific microdeletion syndrome For a child with dysmorphic features, autism spectrum disorders and/or developmental delay For the work up of a child with short stature/failure to thrive once nutritional and endocrine issues have been ruled out Epilepsy work up Don t send: If you suspect a specific single gene disorder (Marfan, NF1, Noonan, etc) If you re not comfortable dealing with the potential of getting a variant of uncertain significance
Interpretation of CMA Reports 1. Normal Female: Arr[hg19] [1-22, X]x2 Male: Arr[hg19] [1-22]x2 [XY]x1 2. Known pathogenic finding
Interpretation of CMA Reports 3. Variant of uncertain significance (VUS)
Interpretation of CMA Reports 4. Loss of heterozygosity (LOH/ROH/AOH) We care about LOH because: 1.Increases the risk for autosomal recessive disorders 2.Tells us about consanguinity (legal/not legal)
You get an abnormal CMA: what next (other than genetics referral)? DiGeorge/22q11.2 microdeletion syndrome: Practical Guidelines for Managing Patients with 22q11.2 Deletion Syndrome. J Pediatr. 2011 August ; 159(2): 332 9 Echocardiogram/cardiology referral Check calcium level, thyroid function Renal ultrasound Hearing/vision evaluations Williams Syndrome: Health Care Supervision for Children With Williams Syndrome. Pediatrics 2001;107;1192 Same as above, plus urine calcium/creatinine Syndrome specific growth charts
You get an abnormal CMA: what next (other than genetics referral)? Variant of uncertain significance: Most, but not all, will not turn out to be clinically relevant Many will end up being inherited from an unaffected parent It would not be wrong to err on the side of assuming that the VUS is not clinically significant when telling the family that something was found Regions of homozygosity: These are not inherently abnormal regions of chromosome, but rather indicate a common ancestor, or sometimes a closer relationship between the parents Assuming that incest is not revealed, the only relevant information this provides is that there is an increased risk for recessive disease, specifically based on what recessive genes are located within these regions In reality, we only figure out that it is one of these genes every once in a while usually, when a genetic etiology is highly suspected, there is another answer
Take Away Points Chromosome Analysis (ideally with extra cell count for mosaicism) should be sent on a child with suspected Down Syndrome or Turner Syndrome FISH has very limited utility in the primary care setting CMAs are first line diagnostic tools in the genetics evaluation for many types of problems, but should not be mistaken as a test that can diagnose single gene disorders/syndromes For most syndromes, while they are waiting for their genetics appointment, you can get a lot of the important evaluations started, which almost always includes cardiac evaluation, renal ultrasound, hearing and vision evaluations, developmental/neurologic evaluations, regardless of diagnosis As it is worse to oversell a VUS as likely pathogenic, it would be better to down play the likely significance of the finding to a family ( this could be nothing, this could be a familial variant ) as we rarely find (or at least rarely feel confident) that the VUS is actually the diagnosis