Genetic Disorders PDF

Genetic disorders Classes of genetic disorders Genetic counselling and testing Online resources Genetic disorders Caused, in whole or in part, by one or more alteration in an individual's genome May be hereditary or acquired during the individual’s lifetime...

Genetic disorders Classes of genetic disorders Genetic counselling and testing Online resources Genetic disorders Caused, in whole or in part, by one or more alteration in an individual's genome May be hereditary or acquired during the individual’s lifetime Germline mutations can be inherited, somatic mutations cannot Three classes monogenic: due to a variation in a single gene chromosomal: due to a change in the number or structure of chromosomes multifactorial/complex: due to alterations in multiple genes (polygenic), often interacting with environmental and lifestyle factors The continuum of genetic disorders For any condition, the overall balance of genetic and environmental determinants can be represented by a point somewhere within the triangle Strachan and Read. HMG5 Fig 5.1 Monogenic diseases A a Due to a variation in a single gene > 6000 disorders A AA Aa Most are rare, affecting 90% manifest before puberty Mostly mendelian inheritance autosomal dominant autosomal recessive X-linked recessive X-linked dominant (few) Y-linked (none) Some maternal inheritance – due to variants in mitochondrial DNA genes Common monogenic diseases in Europe Autosomal dominant familial combined hyperlipidaemia familial hypercholesterolaemia adult polycystic kidney disease Huntington's disease neurofibromatosis myotonic dystrophy Autosomal recessive Marfan syndrome hemochromatosis cystic fibrosis alpha-1-antitrypsin deficiency phenylketonuria spinal muscular atrophy X-Linked recessive sickle cell anaemia fragile X syndrome thalassaemia Duchenne muscular dystrophy X-linked ichthyosis haemophilia A/B The prevalence of inherited diseases varies between populations Example: global distribution of sickle cell disease Kato et al. Nat Rev Dis Primers. 2018;4:18010. Multifactorial or complex disorders Determined by the interaction of multiple genetic factors, in combination with environmental and lifestyle factors Polygenic interplay of variants in many genes, each exerting a small additive effect individuals inherit an increased risk of developing a disease (susceptibility) familial aggregation without a clear-cut inheritance pattern Include pediatric and adult disorders congenital defects: cleft lip/palate, neural tube defect … acquired diseases: diabetes, Crohn’s disease, asthma, autism, hypertension... Note: cancer is multifactorial – always genetic, sometimes hereditary with mendelian inheritance pattern Chromosomal disorders Karyotype analysis: trisomy 13 Due to chromosomal abnormalities occurring during gametogenesis or following fertilization Numerical abnormalities polyploidy aneuploidy Structural abnormalities structural breakage with subsequent reunion in a different configuration deletion, insertion inversion, translocation Common cause of infertility, miscarriage, congenital malformations and intellectual disability Present in 1 in 200 live births FISH: chr.7 translocation Age of expression of genetic disorders Gelehrter, Collins and Ginsburg. Principles of medical genetics. 2° ed. 1998 Impact of genetic disease at different stages of life Before birth: chromosomal abnormalities are present in at least 10% of all recognized conceptions 2% of pregnancies in women >35 years 40-50% of miscarriages in 1st trimester Newborn infants: major congenital anomalies are present in up to 3% of all neonates, of which 50% caused exclusively or partially by genetic factors cause 20-30% of all infant deaths in developed countries; 2nd most common cause of infant death after immaturity-related conditions Source: Emery’s Elements of Medical Genetics and Genomics. 16° ed. 2022 Elsevier Impact of genetic disease at different stages of life Childhood: genetic disorders account for >50% of visual impairment, hearing loss and intellectual disability 30% of childhood hospital admissions in industrialized countries 40-50% of all childhood deaths in developed countries; 2nd most common cause of death after accidents Adulthood: by age 15y, 5% of the population will have a disorder in which genetic factors play an important role >50% of older adult population in developed countries will have a medical problem with agenetic component 5-15% of common cancers (breast, ovarian and colon cancer) have a strong hereditary component Source: Emery’s Elements of Medical Genetics and Genomics. 16° ed. 2022 Elsevier Clinical genetics Provide a diagnostic service and genetic counselling for individuals or families with, or at risk of, conditions which may have a genetic basis. Genetic counselling Clinical and Genetic test family history Diagnostic laboratory Genetic counselling Patients or relatives, at risk of an inherited disorder, are advised of: the nature and consequences of the disorder the risks and benefits of genetic testing the probability of developing or transmitting it options for the management of the disorder Reasons for referral Preconceptional genetic disorder present or suspected in family history of repeated miscarriages assisted reproduction Prenatal history of previous child with birth defect, developmental delay, or other genetic condition carriers of recessive condition abnormal ultrasound or maternal serum screening results → risk of aneuploidies and neural tube defects Postnatal (infancy to adulthood) newborn with multiple birth defects or positive neonatal screening results child with developmental delay, sensory impairment, intellectual disability, or other suspected genetic condition adult with suspected adult-onset disease Oncologic familial aggregation of cancer  Newborn screening for hereditary diseases Purpose: early identification of diseases that cause permanent disability or death if not identified and treated as early as possible. Biochemical assays for specific serum analytes: tandem mass spectrometry, immunoassays, enzyme assays … Identifies newborns at risk, confirmed with diagnostic molecular tests Newborn screening in Italy: Mandated by Articolo 6, legge 104, 05/02/1992; Decreto del Ministero della Salute, 13/10/2016 49 diseases screened nationally including cystic fibrosis, congenital hypothyroidism, phenylketonuria (PKU), and other metabolic diseases https://www.osservatorioscreening.it Large-scale genomic newborn screening studies launching internationally Cohort sizes and proposed screening approaches are shown. TBD, to be determined; WGS, whole- genome sequencing. Stark and Scott. Nat Rev Genet. 2023 Jun 29. doi: 10.1038/s41576-023-00621-w. Epub ahead of print. The genetic counselling process 1. Establishing the diagnosis a. Gathering information personal and family clinical history, even of those deceased photographs b. Reconstructing the pedigree diagram showing family relationships extended over at least 3 generations (if possible): proband, parents, grandparents c. Specialist visits ophthalmolgy, cardiology, neurology etc. d. Genetic testing informed consent cytogenetic and/or molecular testing The genetic counselling process 2. Risk assessment risk of developing disease (if not already manifesting) risk of recurrence: probability that an inherited disease present in a family will recur in another member of that family based on pattern of inheritance, genetic nature of disease and empirical data 3. Communication and support communicates information and test results to proband and/or proband’s family refers to specialist centers for disease management directs towards patient support groups Constructing family pedigrees Proband propositus = the person through whom a family is being ascertained  Genetic testing Genetic test: laboratory test used to identify genetic alterations associated with a disease. Molecular diagnostics PCR-based assays: PCR, restriction analysis (RFLP), allele-specific PCR (ARMS), oligonucleotide ligation assay (OLA), multiplex-ligation dependent probe amplification (MLPA), real time quantitative PCR (qPCR), digital droplet PCR (ddPCR) hybridization-based assays: reverse dot blot, fluorescence in situ hybridization (FISH) array-based assays: array comparative genomic hybridization (aCGH), SNP arrays sequencing technologies: Sanger sequencing, next generation sequencing (NGS) other methodologies: MALDI-TOF mass spectrometry Cytogenetic testing karyotyping/chromosomal banding  The purpose of genetic testing (1) Diagnostic: to confirm a diagnosis when a genetic condition is suspected symptomatic individuals newborn with positive biochemical screening results Prenatal: to detect a genetic condition before birth high risk pregnancies: family history of genetic condition, advanced maternal age, history of miscarriages/stillbirths/birth defects, carriers for recessive conditions, positive maternal screening results Preimplantation: to detect genetic defects in embryos created through in vitro fertilization (IVF) to detect specific monogenic conditions (e.g., for carriers of a recessive condition) to screen for aneuploidies (advanced maternal age, history of miscarriages/stillbirths/birth defects)  The purpose of genetic testing (2) Carrier screening: to identify healthy carriers of genetic variants causing recessive conditions healthy relatives of affected individuals general population (e.g., Israeli carrier screening program for reproductive purposes for the prevention of Tay-Sachs disease, thalassemia, cystic fibrosis and other hereditary diseases) Predictive: to predict future risk of disease in an asymptomatic person with a family history of disease presymptomatic: eventual development of symptoms is certain when the gene variant is present (e.g., Huntington disease) predispositional/susceptibility: eventual development of symptoms is likely but not certain when the gene variant is present (e.g., BRAC1/BRAC2 in hereditary cancer) Pharmacogenetic: to detect genetic variants that are associated with variable drug responses, and tailor therapy to the individual’s genetic profile  Genetic testing in clinical settings A genetic test is normally performed just once, and the result forms a permanent part of a person’s health record. When a clinician requests a diagnostic genetic test, there are three possible questions: Does the patient have a specific variant in a particular gene that can explain the disease? Does the patient have any variant in this particular gene that might cause the disease? Does the patient have any variant anywhere in the genome that would explain the disease? Genetic testing has to be tailored to the condition and suspected genetic mechanism Genetic testing strategies Specific variant: detect a specific variant known to be 1 base responsible for a particular disorder Single gene: test a single gene for potentially causative 1 gene variant(s), appropriate for disorders with allelic heterogeneity1 Gene panel: test for causative variants in many genes in a A c.1138G>C (98%) (1%) Fig 9.20 Emery’s Elements of Medical genetics, 15ed Image modified from: Vajo et al.. Endocr Rev. 2000 Feb;21(1):23-39 Testing for multiple variants in a single gene Example: CHARGE syndrome rare congenital disorder clinical features: Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and development, Genitourinary abnormalities, and Ear anomalies and deafness, plus others … causative gene: CHD7 - only gene identified so far > 500 pathogenic variants (https://www.chd7.org/) nonsense, frameshift, missense, splice site, synonymous, exon deletion/duplication, gene deletion methodology: targeted NGS of CHD7 gene Figure: Hefner & Fassi. Am J Med Genet C Semin Med Genet. 2017 Dec;175(4):407-416. Testing for variants in a panel of genes Example: RASopathies group of developmental syndromes caused by variants in genes of RAS-MAPK signalling pathway clinical features: distinct facial features, developmental delays, cardiac defects, growth delays, neurologic issues, and gastrointestinal difficulties methodology: NGS gene panel targeting coding sequences of 32 RAS-MAPK genes https://rasopathiesnet.org/2011-symposium-abstracts/rasopathies-horiz2-1-2/ Note: NGS gene panels allow simultaneous testing of multiple clinically- and genetically-related conditions When is exome sequencing useful? when a disorder is suspected to be genetic but is not recognizable clinically (e.g., birth defects and developmental delay) disease mechanism is poorly understood; causative gene is unknown when the patient's symptoms are consistent with a wide range of genetic disorders avoids the use of multiple gene panels → more cost-effective when first-line tests have failed to identify causative variants can uncover relevant variants in genes not previously implicated in a given disease Multiple tiers of testing Definitive molecular diagnosis may require multiple tiers of testing, Tier 1 even when the clinical phenotype is NO clear, and the disease-associated gene is known Tier 2 NO Tier 3 NO Tier 4 Cystic fibrosis CFTR gene >2500 known variants; >300 pathogenic variants Most common variant Phe508del, present in 60% of affected individuals (20% homozygotes, 40% heterozygotes) CFTR gene Image modified from Tsui and Dorfman. Cold Spring Harb Perspect Med. 2013;3(2):a009472.  Multiple tiers of testing: the example of cystic fibrosis (CFTR gene) Società Italiana di Genetica Umana (SIGU) guidelines (https://sigu.net/category/linee-guida-e-raccomandazioni/) "Consensus per l’analisi genetica in fibrosi cistica", 2019 Tier Purpose Target Methods Detection rate Level 1 Test for the most common 50-80 variants (SNVs and small Reverse dot-blot, ARMS, 85% pathogenic CFTR variants in indels), including Phe508del MALDI-TOF mass the population spectrometry, NGS variant panels Level 2 Scan the CFTR gene for Variants (SNVs and small indels) Sanger sequencing, 95% potentially causative in exons, intron/exon junctions, NGS (including variants variants promoter, 3’UTR, known intronic detected by level 1) splice variants, polyT/TG tract Level 3 Detection of Exon deletion/duplication MLPA, real-time PCR, aCGH 2%, macrodeletions and 97% final detection rate macroduplications for levels 1+2+3 Level 4 Detection of unknown Alternatively spliced mRNA RT-PCR followed by 1%, intronic splice variants sequencing of cDNA 98% final detection rate for levels 1+2+3+4 SNV = single nucleotide variant; indel = insertion and/or deletion In recent years, NGS has revolutionized the diagnosis of genetic diseases! Clinical classification of variants ACMG Standards and Guidelines (2015) Five tiers of variant classification for mendelian diseases based on weight of evidence Pathogenic: causative of disease Likely pathogenic: likely causative 90% certainty) Variant of uncertain significance (VUS) Likely benign: likely not causative (90% certainty) Benign: not causative of disease Richards et al. Genet Med. 2015 May;17(5):405-24. Online resourses: Online Mendelian Inheritance in Man Catalog of inherited disorders McKusick-Nathans Department of Genetic Medicine, Johns Hopkins Medicine (USA) https://www.omim.org Clinical Gene synopsis (indicated with asterisk * before number) OMIM search Example: Inherited diseases of the thyroid gland *thyroid* → 1301 entries *thyroid* NOT *parathyroid* → 1040 entries # Inherited phenotype: #1 Autosomal (pre-1994) #2 Autosomal (pre-1994) #3 X-linked #4 Y-linked #5 Mitochondrial #6 Autosomal (post-1994) % Mendelian phenotype or phenotypic locus, molecular basis unknown No symbol before number → mendelian basis suspected but not clearly established ORPHANET: the portal of rare diseases and orphan drugs Languages: english, french, spanish, german, italian, portuguese, dutch http://www.orpha.net GeneReviews – NCBI Bookshelf Example: achondroplasia https://www.ncbi.nlm.nih.gov/books/NBK1116/ Reading Strachan and Read. Human Molecular Genetics 5th edition, 2018. Chapter 20 Genetic testing in healthcare and the law Thompson and Thompson. Genetics in Medicine, 8th edition, 2016 Chapter 16 Risk Assessment and Genetic Counseling