Basic Genetics Daniela Iacoboni, MS, CGC Genetic Counselor Michigan State University Department of Pediatrics and Human Development September 1, 2010 Chromosome Structure and Analysis Chromosome Structure Chromatids Telomeres Short arm p Satellite Stalk centromere Long arm q Metacentric Submetacentric Acrocentic Types of Abnormalities • Numerical – Aneuploidy, euploidy • Structural – Rearrangements between or within chromosomes • Epigenetic – Difference in processing of genetic material – Causes change in gene expression but is not due to DNA sequence alterations – Imprinting and uniparental disomy (UPD) Effects of Abnormalities • Dosage effect – deletion or duplication of all or part of a chromosome • Damaging effect – disrupt gene at breakpoint of a rearrangement • Positional effect – gene in a new chromosome “environment” behaves differently • Parent of origin effect – genomic imprinting Aneuploidy: Meiotic Errors • Most nondisjunction occurs in maternal meiosis I – 47,XYY typically paternal meiosis II error – ~50% 47,XXY are paternal meiosis I errors – 45,X typically absence of paternal sex chromosome contribution Aneuploidy Aneuploidy: Maternal Age • • • • Poor “quality control” in egg cells compared to sperm Increased disorganization at meiosis with older maternal age Lower number of remaining eggs in ovaries Changing extra-ovarian environment Aneuploidy: Parental Predisposition? • ? Some individuals prone to nondisjunction? • Recurrence risk estimates based on data from Warburton et al 2004 (measured at amniocentesis): Index pregnancy Same trisomy Other viable trisomy Trisomy 21, maternal age <30 8.2 x age risk* 2.5 x age risk Trisomy 21, maternal age >30 2.1 x age risk 2.3 x age risk Other viable trisomy 1.7 x age risk Nonviable trisomy 2.4 x age risk 1.8 x age risk *For practical purposes, recurrence risk for a woman who has had a prior trisomic pregnancy, recurrence risk is overall 1.6-1.8 times her age-related risk Aneuploidy: Mitotic Errors • Mitotic nondisjunction is primary cause of mosaicism (e.g. 46,N/47,N,+21) – Normal OR abnormal zygote – Trisomic, monosomic and normal cell line Aneuploidy: Mosaicism in Prenatal Diagnosis • Cell culture can cause artifactual mosaicism • Confined placental mosaicism – One or more cell lines detected only in placental tissue but not fetal tissue – THIS IS NOT ALWAYS BENIGN (additional analysis required) • Maternal cell contamination is most common cause of 46,XX/46,XY mosaicism – MTC studies can confirm – Note: 46,XX result in male fetus may be true sex discrepancy (should review sonogram and r/o sample mixup) Numerical Abnormalities: Euploidy • Triploidy (3n) – Dispermy most common cause – Also caused by failure of meiosis I or II by egg or sperm – 99% lost by 2nd trimester 69,XXY • Cause of 1-3% recognized miscarriages • Tetraploidy (4N) very rare Structural Abnormalities • Balanced – normal amount of chromosome material but rearranged • Unbalanced – missing or extra chromosome material • Most de novo rearrangements occur in male spermatogenesis • Rarely, errors in mitosis will result in mosaicism for a structural abnormality Structural Abnormalities: Deletions • Loss of chromosome segment • Phenotype depends on size and location of deletion • May be terminal or interstitial • Subtelomeric deletions – Account for 3% of cases of mental retardation (with normal karyotype) • Microdeletion syndromes – Most detected only on FISH (array CGH) – Recurrent and typically de novo Structural Abnormalities: Duplications • Results in partial trisomy • In general, less clinical severity Structural Abnormalities: ESACs • Extra Structurally Abnormal Chromosomes (ESACs) – Also called marker or supernumerary chromosomes – Duplication (partial trisomy) or triplication (partial tetrasomy) of material • Difficult to classify – May or may not cause phenotypic effect • If marker is noted on prenatal diagnosis, test parents Structural Abnormalities: Isochromosomes • Isochromosome is a mirror image marker – Two identical arms on either side of centromere – Results in tetrasomy of segment involved – 46,X,i(Xq) or 45,X/46,X,i(Xq) accounts for 18% Turner syndrome Structural Abnormalities: Insertions • Additional material inserted into a chromosome – Interchromosomal insertion • Most common • Segment from 1 chromosome inserted into another – Intrachromosomal insertion • Very rare • High reproductive risk – Average risk for abnormal child: • 32% if male is carrier • 36% if female is carrier Structural Abnormalities: Inversions • Pericentric – Breakpoints include centromere • Paracentric – Breakpoints do not include centromere • Several benign variants: – Heterochromatic regions around centromere of chromosomes 1, 9, 16, Y – Many difficult to detect • Recognized in <1% of individuals • Reproductive risks depends on nature of inversion (GC) Structural Abnormalities: Rings • Uncommon • 99% are sporadic • Most cause abnormal phenotype – Dysmorphology – Mental retardation – Short stature • 45,X/46,X,r(X) accounts for 16% Turner syndrome Structural Abnormalities: Reciprocal Translocations • Balanced translocations – – • Exchange of material between 2 nonhomologous chromosomes No apparent gain or loss Usually no phenotypic effects in balanced carrier – – Break within a gene could result in phenotype Could have deleted material not visible with standard cytogenetics (CGH array) • Balanced carriers have risk for unbalanced offspring • De novo, apparantly balanced translocations detected prenatally, 6% risk of adverse fetal outcome (additional 3% risk above general population risk) Structural Abnormalities: Robertsonian Translocations • Fusion of two acrocentric chromosomes (13, 14, 15, 21, 22) • Most involve chromosomes 13 and 14 Cytogenetic Nomenclature Epigenetic Effects: Imprinting • Before fertilization, parts of the genome are “marked” as maternal (from egg) or paternal (from sperm) • Imprinting must be reset in germ cells • Differences in gene expression depending on parent of origin • Mechanism not entirely understood but involves DNA methylation Epigenetic Effects: Imprinting and UPD • UPD-- Inheritance of all or part of both chromosomes in a pair from a single parent – 6, 7, 11, 14 and 15 • Occurs by a number of mechanisms • Recurrence risk not known to be increased • Disorders caused by UPD: – – – – PWS (25% maternal UPD 15) AS (7% paternal UPD 15) BWS (10% paternal UPD 11) Russell-Silver syndrome (7-10% maternal UPD 7) – Transient neonatal diabetes (? 50% paternal UPD 6) • Can result in autosomal recessive disorder when only 1 parent is a carrier Epigenetic Effects: Human Imprinting http://genes.uchicago.edu/upd/ Genes • Consist of a long combination of four different nucleotide bases • Many possible combinations of these four nucleotides: – A (adenine) – C (cytosine) -T (thymine) - G (guanine) • Amino acids are coded by different trinucleotide base combinations Anatomy of a Gene Stop codon Mutations • Involve large or small DNA alterations • Point mutation-change in a single base pair • May produce one of three types of mutations: missense, nonsense, frameshift Point Mutations: Missense • One amino acid is substituted for another • May or may not alter amino acid code • Even if code is altered, it is not necessary deleterious Point Mutations: Nonsense • Nonsense mutation-stop codon leads to premature termination of translation Point Mutations: Frameshift • Causes a change in the reading frame – leads to introduction of unrelated amino acids into the protein • Small deletions have effects similar to frameshift mutations • 1/3 do not alter the reading frame • Results in removal of a small number of contiguous amino acids Nucleotide Repeats • Normal di, tri, tetra nucleotide repeats • Found in multiple sites in the genome – Mostly benign – Repeat number varies between individuals – Have been used as genetic markers for gene mapping by linkage, paternity testing, forensics – Not completely stable – number of repeats can change up or down • Larger insertions and deletions – In non coding regions (introns), may have little or no effect – In coding or control regions (exons), may disrupt gene function • Problem in gene therapy Gene Duplications/Deletions • Arises by unequal crossing at regions containing homologous copies of a DNA sequence Deletion • • • Duplication Duplication of whole or part of gene Probable mode by which “new” genes have evolved Genome is littered with “pseudogenes” • Homologous sequences which are not transcribed or not translated Family History and Inheritance Patterns Common Pedigree Symbols Autosomal Dominant Autosomal Recessive X-Linked Inheritance Mitochondrial Genetics • Circular DNA in mitochondria – • • • Believed to have prokaryotic origin (parasitic) Mutations in mitochondrial genome lead to a variety of diseases involving muscle, brain, and liver (among other high energy and metabolism dependent tissues) : – LHON, MELAS, MERRF,NARP, diabetes, hearing loss Often, mutations are acquired Mutations can be inherited – Always from mother (oocyte) – All mitochondrial DNA (mtDNA) comes from the egg, including any mutations – Not all the DNA is mutated, so as the fertilized egg multiples, there is a mix of DNA (mosaic) – The percent of mutated mtDNA vs unaffected DNA will vary Genetic Testing • Cytogenetics – numerical and structural anomalies – i.e., karyotyping • Molecular genetic studies – single-gene disorders – i.e., gene sequencing • FISH (fluorescence in situ hybridization) – can assess for deletions/duplications undetectable on routine cytogenetics – prenatal assessment for aneuploidy (13,18,21,X,Y) Future Directions - CGH • Comparative Genomic Hybridization (CGH) • Combines molecular techniques with cytogenetics – Detects submicroscopic duplications and deletions • Fluorescently labeled patient DNA hybridizes to array • Measure differences in fluorescence • Current resolution is 6 to 35 kb ©2007 Signature Genomic Laboratories, LLC ©2007 Signature Genomic Laboratories, LLC ©2007 Signature Genomic Laboratories, LLC Karyotype vs. Array In Unexplained DD/ID, ASD, MCA Karyotype: Array: • 3% diagnostic yield • 3% diagnostic yield on subtelomere FISH • 15-20% diagnostic yield Utility of CGH • Benefits: – Whole genome coverage: you don’t have to know what you are looking for – Menten (2006): imbalances in 8% of individuals with normal karyotype and subtelomeric screening • Limitations: – Requires significant data manipulation, complex algorithms – Cannot detect balanced abnormalities (translocations, etc) – Many polymorphic areas in human genome – not all identified “abnormalities” are truly abnormal • Requires sample from both parents to determine if de novo • Still may not be able to confirm identified anomaly is cause of features – In many cases, limited or no clinical information available – Uncertain sensitivity in detecting mosaicism Consensus Statement • Am J Human Genetics 5/2010 – Array as first-tier cytogenetic test for patients with DD/ID, ASD, MCA – Karyotyping reserved for: • Patients with obvious chromosomal syndromes • Family history of chromosomal rearrangement • History of multiple miscarriages Prenatal Array • High sensitivity and specificity for detection of clinically significant unbalanced abnormalities • Minimizes detection of CNVs of uncertain clinical significance • Parental bloods required – Currently, prenatal CGH panels avoid certain adult-onset disorders • violates ethical principles of genetic testing (issues of counseling and informed consent) ACOG Committee Statement • November 2009 • Array is NOT a replacement for classic karyotype in prenatal diagnosis – Can be offered as adjunct tool in prenatal cases with abnormal anatomic findings and a normal conventional karyotype – Recommend pre- and post-test counseling – May be a useful screening tool in the future, but more studies are necessary Important Lessons • No single test or technology can “rule out” all chromosome abnormalities • Technology is quickly evolving • Currently available technologies may be obsolete in just a few years Vignette 1: Prenatal Case Presentation A 21-year-old woman was referred at 16 weeks’ gestation because alobar holoprosencephaly (HPE) was observed on ultrasound examination. The woman’s obstetrical history revealed one spontaneous abortion and one pregnancy terminated at 15 weeks’ gestation following detection of alobar HPE. Family history showed that the father’s brother had failure to thrive and autistic features. Karyotype analysis on the mother and fetus was normal. Vignette 1: Prenatal Case Presentation • Microarray analysis of the fetus showed: – single copy-number loss of 7.7Mb at 7q36.1q36.3 encompassing the SHH gene, deletion of which is associated with holoprosencephaly 3. – single-copy gain of 4.4Mb at 8q24.3. FISH confirmed both abnormalities. – FISH analysis of the parents showed a balanced t(7;8)(q36.3;q24.3) in the father. Vignette 2: Prenatal Case Presentation • 35-y.o. woman referred for AMA • Amnio finds 13;14 robertsonian translocation • What is the next step? • Parental studies • Father has same translocation • Are we done? Vignette 2: Prenatal Case Presentation • Maternal UPD 14 – premature birth – slow growth before and after birth – short stature – developmental delay – small hands and feet – early onset of puberty • Paternal UPD 14 – excess amniotic fluid – an opening in the wall of the abdomen – distinctive facial features – a small, bell-shaped chest – short ribs – developmental delay Case Example 1: 1p36 Deletion • Mental retardation • Seizures • Structural neurological anomalies • Delayed closure of ant. fontanelle • Hearing loss • Vision and oculomotility • Poor/absent speech • Behavioral problems Case Example 2: 16p11.2 Deletion •Autism/ASD •Developmental delay •Psychiatric disorders •Behavioral problems •Hyperactivity •Mild to moderate MR •Overweight/Obesity •Mild dysmorphic features Case 3: 22q11.2 Deletion • • • • Heart defects Hearing loss Cleft palate Velopharyngeal insuff • Learning disabilities • Immune deficiency • Renal anomalies Case 4: 22q11.2 Duplication • • • • • Heart defects Hearing loss CI/DD Velopharyngeal insuff Urogenital • Psych/behavioral References • • • • • • • • • • Gardner RJM and Sutherland GR. (2004) “Chromosome Abnormalities and Genetic Counseling.” Oxford University Press, New York Genetic Counseling Aids (2002) Greenwood Genetic Center. Lapierre JM and Tachdjian G (2005) Detection of chromosomal abnormalities by comparative genomic hybridization. Current Opinion in Obstetrics and Gynecology 17:171-177. Menten B et al. (2006) Emerging patterns of cryptic chromosomal imbalance in patients with idiopathic mental retardation and multiple congenital anomalies: a new series of 140 patients and review of published reports. Journal of Medical Genetics 43:625-643. Niemitz EL and Feinberg AP (2004) Epigenetics and assisted reproductive technology: a call for investigation. American Journal of Human Genetics 74:599-609. Signature Genomics Laboratories, LLC. www.signaturegenomics.com Rosenberg C et al. (2006) Array-CGH detection of micro rearrangements in mentally retarded individuals: clinical significance of imbalances present both in affected children and normal parents. Journal of Medical Genetics 43:180-186. University of Washington Human Ideogram Album (August 2006) http://www.pathology.washington.edu/research/cytopages/idiograms/human/hum_01.pdf University of Chicago Human Imprinting Map (August 2006) http://genes.uchicago.edu/upd/ Warburton D, et al. (2001) Trisomy recurrence: a reconsideration based on North American data. American Journal of Human Genetics 69S: 232.
