General Nuts and Bolts of Genetics

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1 General Nuts and Bolts of Genetics Michael J. Gambello, MD, PhD Section Chief, Division of Medical Genetics Primer in Genetics Chromosomes: 46, XX or 46, XY. 23 chromosomes from mother, 23 from father. Mitochondrial DNA Genes arranged on chromosomes which code for proteins (enzymes, transporters, collagens etc). The Double Helix April 1953 James Watson Francis Crick Rosalind Franklin Maurice Wilkins

Replication Mechanism It has not escaped out notice that

suggests a possible copying mechanism for the genetic

Crick A C T G Chromosomes are DNA Each Chromosome really

2 Replication Mechanism It has not escaped out notice that The specific pairing we have postulated Immediately suggests a possible copying mechanism for the genetic material. J.D. Watson and F.H.C. Crick A C T G Chromosomes are DNA Each Chromosome really One Molecule of DNA wrapped around Proteins - Histones The Genetic Code Room for Normal Variation

Transfer of Biological Information Gene String of A s, T s C s and G s ATG GGG

AUG GGG UUU UCU CCA CAC Met Gly Phe Ser Pro His Codons Enzyme Transporter etc.

of the American Academy of Nutrition and Dietetics 114(2):299-312 Children s

3 Transfer of Biological Information Gene String of A s, T s C s and G s ATG GGG TTT TCT CCA CAC TAC CCC AAA AGA GGT GTG mrna (U instead of T Single Stranded) AUG GGG UUU UCU CCA CAC Met Gly Phe Ser Pro His Codons Enzyme Transporter etc. Met Met Gly Ser Pro Phe Pro His His Genes are Come in Pieces - Exons Journal of the American Academy of Nutrition and Dietetics 114(2): Children s February Healthcare 2014, DOI: /j.jand of Atlanta Source: PubMed

Genetic Disease Mutations in DNA Genetic Disease Single base variants Other Mechanisms: Small Gene

Rearrangements Epigenetic changes GENETIC TESTING TARGETS THE LIEKLY MECHANISMS OF DISEASE The Age

stuff in between Genomics: Study of all genes and their interactions Proteome: All the proteins

4 Genetic Disease Mutations in DNA Genetic Disease Single base variants Other Mechanisms: Small Gene Deletions Whole Gene Deletions Repeat Disorders Whole Chromosome defects Chromosomal deletions Rearrangements Epigenetic changes GENETIC TESTING TARGETS THE LIEKLY MECHANISMS OF DISEASE The Age of Omics Suffix Ome = Collective study of Pools of Biological Molecules Genome: All the genes, and stuff in between Genomics: Study of all genes and their interactions Proteome: All the proteins Proteomics: Study of all proteins and their interactions Metabolome: All the metabolites, small molecules etc.

1% of genome codes for protein The EXOME (all the Exons ) Genes are scattered non randomly across genome.

5 Human Genome Project First Genomic Project International Effort Completed 2003 To understand the human genome. Determine the 3 billion A s, T s, G s and C s Map all our genes Knowledge from HGP Completed in Only ca. 1% of genome codes for protein The EXOME (all the Exons ) Genes are scattered non randomly across genome. Only 22,000 genes (much less than we thought). Genetic Testing Karyotype vs. Chromosome Microarray Repeat Disorders Next Generation Gene Panels Whole Exome vs Whole Genome Sequencing

6 Whole Chromosome Aneuploidies Rudolph s Brief Atlas of the Newborn, 1998 G Banded Karyotypes Lymphocytes or amniocytes Only Used for Recognizable WHOLE chromosome syndrome (21, 13, 18, 47, XXY etc) Couples with recurrent miscarriage looking for balanced rearrangements Translocation Down Syndrome Translocation Down syndrome ~ 4% cases due to 21 translocated to another chromosome 46,XYder(13;21)(q10;q10)+21 MUST KARYOTYPE PARENTS!!

Copy number variants = CNVs Small pieces of

7 Chromosome Microarray Can detect small deletions of duplications in the genome. Copy number variants = CNVs Small pieces of entire genome placed on glass slide. Using UCSC Genome Browser (Human Genome Project) we know exactly what genes are deleted or duplicated. Replaces traditional Karyotype Much better resolution 5 Mb vs 30 kb Microarray Chromosome Microarray

Our recommendation based on current evidence is to

of G banded karyotype, for patients with unexplained

A consensus statement from the: International

Microarray Results Dynamic Mutation Diseases caused

8 Our recommendation based on current evidence is to offer CMA as the first tier genetic test, in place of G banded karyotype, for patients with unexplained DD/ID, ASD, or MCA. A consensus statement from the: International Standards for Cytogenomic Arrays (ISCA) Consortium Microarray Results Dynamic Mutation Diseases caused by Repeats: CCCCGCCCCGCG EPM1 SCA8 SCA12 SCA17 Adapted from Mirkin Nature 447, (2007).

Fragile X Syndrome http://medicineworld.

gif FMR1 Molecular Genetics DNA Normal FMR1 Allele Transcription

http://www.sueblimely.com/images/2008/fragilex-fmr1-protein.

9 Fragile X Syndrome FMR1 Molecular Genetics DNA Normal FMR1 Allele Transcription (CGG) 6-44 ATG mrna (CGG) 30 AUG Translation FMR1 PROTEIN Males with Fragile X Syndrome DNA CH 3 (CGG) 200 Transcription Factors Cannot Bind ATG No mrna Production NO FMR1 Protein Production

Phenotype NOT Consistent So for any Boy with intellectual Disability order FMR1

html Single Gene Sanger Sequencing Hereditary Fructose Intolerance Patient

CCC UUU UUC CUC UGA ATG CTC CCC TTT TTC CTC TGA 1 Chromosome Microarray CANNOT

10 Phenotype NOT Consistent So for any Boy with intellectual Disability order FMR1 CGG Repeat Analysis Single Gene Sanger Sequencing Hereditary Fructose Intolerance Patient Homozygous mutation in ALDOB gene c.448g>c (p.a150p) Missense c.448g>c (p.a149p) Proline Coding DNA protein A of ATG start is 1 Alanine mrna cdna AUG CUC CCC UUU UUC CUC UGA ATG CTC CCC TTT TTC CTC TGA 1 Chromosome Microarray CANNOT DETECT Point mutations Repeat Disorders Very small intragenic deletions or duplications (e.g. Cystic Fibrosis c del CTT = p.phe508del [aka deltaf508) Balanced Translocations

Genomics in Medicine Based on Next Generation Sequencing Technology Gene Sequencing Panels Whole Exome vs Whole Genome Sequencing Gene Panels Can interrogate MANY genes with one test.

11 Genomics in Medicine Based on Next Generation Sequencing Technology Gene Sequencing Panels Whole Exome vs Whole Genome Sequencing Gene Panels Can interrogate MANY genes with one test. Very useful with disorders that are genetically heterogeneous (MANY genes) Genetically heterogeneous diseases: Deafness, Epilepsy, Cardiomyopathy Whole Exome Sequencing Sequence MOST (92 96%) EXONS (1% genome protein coding regions Better coverage for known Mendelian disease genes Cost varies $ Sequencing getting cheaper analysis costly Diagnostic Yield 20 30%

WES and Undiagnosed Disease Performed mainly for patients with suspected genetic disease and without a clear clinical diagnosis or those having negative test results for genes known to be associated

Gene for Kabuki syndrome WES of 10 unrelated individuals Genotype Phenotype stratification Identified mutations in MLL2 Clinical testing for Kabuki syndrome now available New Disease Genes Every

12 WES and Undiagnosed Disease Performed mainly for patients with suspected genetic disease and without a clear clinical diagnosis or those having negative test results for genes known to be associated with the disorder. Many examples of novel disease discovery or atypical manifestation of known disease. Gene for Kabuki syndrome WES of 10 unrelated individuals Genotype Phenotype stratification Identified mutations in MLL2 Clinical testing for Kabuki syndrome now available New Disease Genes Every month!! Study of autosomal recessive osteogenesis imperfecta in Arabia reveals a novel locus defined by TMEM38B mutation. Mutations in ATP1A3 Cause Alternating Hemiplegia of Childhood. Two novel CCDC88C mutations confirm the role of DAPLE in autosomal recessive congenital hydrocephalus. Whole Exome Capture and Sequencing Identifies HEATR2 Mutation as a Cause of Primary Ciliary Dyskinesia. CSF1R mutations identified in three families with autosomal dominantly inherited leukoencephalopathy.

13 Successes with Gene Panels or WES CASE 1 15 yo and 22 yo siblings with RP and epiplepsy. Eye disorders panel. Diagnoses with Neuronal Ceroid Lipofuscinosis CLN8 CASE 2 19 yo with PKU but learning and behavior deficits not consistent with PKU. WES identified mutation EHMT1 causing Kleefstra syndrome CASE 3 21 yo with Learning disabilities, dysmorphic, short stature, brachydactyly. WES identified SMAD4 mutation causing Myhre syndrome. Risk for pericardial effusion (once with no FU). Now followed closely by cardiology. Incidental Findings: Exome Sequencing 0 5 incidental finding per exome: - Carrier status of autosomal recessive conditions - Adult-onset conditions 0 2 pharmacogenetic findings per exome Most common variants associated with: Hemochromatosis G6PD deficiency Incidental Findings Consent process very important. Patients can opt out of receiving information Important to have Clinical Genetics Involved inpatient or outpatient

Genomics Where are we Going? Whole Genome Sequencing?

Pharmacogenomics Warfarin and CYP2C9 and VKORC1 genotypes CHALLENGE: Demonstrate Clinical Utility Newborn Screening and

??? Several Studies underway Many issues that need to be addressed.

14 Genomics Where are we Going? Whole Genome Sequencing??? RESEARCH ONLY NOW Personalized Medicine Primary Care DNA vs Family history Predictive Health Targeted Cancer Therapies Pharmacogenomics Warfarin and CYP2C9 and VKORC1 genotypes CHALLENGE: Demonstrate Clinical Utility Newborn Screening and Genomics? NBS based mainly on analyte analysis. MS/MS WES or WGS for NBS???? Several Studies underway Many issues that need to be addressed. NBS and WES or WGS The significance of many variants unknown. GENOTYPE vs. PHENOTYPE Cost still too prohibitive. Exon capture not 100%. What do you do with Adult onset diseases? Do you report carrier status? Do you only report some information in newborn period, and other information after age 18? Ethical, Legal and Social issues.

Resources Gene Reviews https://www.ncbi.nlm.nih.gov/books/nbk1116/ OMIM On Line Mendelian Inheritance in Man https://www.omim.

georgia.gov/nbs-providers Genomic Medicine is here and progressing full speed ahead.

15 Resources Gene Reviews OMIM On Line Mendelian Inheritance in Man Unique The Rare Chromosome Disorder Support Group Newborn Screening Genomic Medicine is here and progressing full speed ahead. There is still a huge amount of work to do to understand our genome and its relationship to health and disease. Goal Improved health for all. Increasing Number of Nuts and Bolts!!!!

CURRENT GENETIC TESTING TOOLS IN NEONATAL MEDICINE. Dr. Bahar Naghavi

2 CURRENT GENETIC TESTING TOOLS IN NEONATAL MEDICINE Dr. Bahar Naghavi Assistant professor of Basic Science Department, Shahid Beheshti University of Medical Sciences, Tehran,Iran 3 Introduction Over 4000