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Genetic tests overview - Karyotype, FISH, MLPA, CMA, Sanger sequencing and NGS

This document provides an overview of common genetic tests, including their indications, techniques, advantages, and limitations. It discusses chromosomal disorders and tests like karyotyping, array comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH), and multiplex ligation-dependent probe amplification (MLPA) that are used to detect chromosomal abnormalities. It also covers Mendelian disorders and gene tests such as Sanger sequencing, next-generation sequencing panels, clinical exome sequencing, and whole exome/genome sequencing that can be used to identify mutations underlying genetic conditions.

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Overview of Common Genetic Tests DR. CHAKSHU CHAUDHRY SENIOR RESIDENT GENETIC METABOLIC UNIT PGIMER 8/6/21
Chromosomes Genes
• “Coloured bodies” • Chromosomes are thread-like structures • Composed mainly of chromatin - that carries highly ordered sequence of linked gene • Resides in Nucleus of Eukaryotic cells • Exist in pairs. Human cells contain 23 such pairs or 46 chromosomes. – Tjio and Levan in 1956 Chromosomes
Chromosomal disorders When to suspect a Chromosomal disorder? / How to identify? What are types of Chromosomal disorders? What are the tests available for testing chromosomal disorders? Indications Technique Advantages Limitations
Antenatal • Antenatally detected cystic hygroma or increased NFT • Multiple Soft markers • +/- Advanced Maternal age When to suspect a Chromosomal disorder? / How to identify? Children • Constellation of clinical features consistent with known disorder • Dysmorphism with or without behavioral problems • Unexplained ID Newborns • Multiple congenital malformations • Significant symmetrical growth restriction and microcephaly • Facial Dysmorphism • Disorders of sexual development
What are types of Chromosomal disorders? NUMERICAL ANEUPLOIDY n=46+/-1 POLYPLOIDY 3n, 4n STRUCTURAL DELETION DUPLICATION INVERSION TRANSLOCATION AUTOSOMES SEX CHROMOSOMES PERIcentric PARAcentric BALANCED UNBALANCED Reciprocal Robertsonian
Inversion PERIcentric PARAcentric Translocation  Reciprocal  Robertsonian
What are the tests available for testing chromosomal disorders? Non Targeted • KARYOTYPE • CMA/aCGH Targeted • FISH • MLPA
Karyotyping Banding Techniques FISH MLPA Comparative Genomic Hybridization
• Karyotype: A chromosome complement of a cell or species • Karyogram / Ideogram : A pictorial display of metaphase chromosomes from a mitotic cell • Cytogenetics: Study of chromosomes and inheritance • Molecular Cytogenetics: Techniques that deal with the entire genome or specific DNA sequences to analyse genomic structural and behavioural variations at chromosomal or subchromosomal level Karyotype and Cytogenetics
Procedure of Karyotyping
Karyotype is based on - Size - Centromeric position - Banding pattern Large metacentric:1-3 Large submetacentric : 4-5 Medium Submetacentric : 6-12 + X Large Acrocentric : 13-15 Small Submetacentric : 16-18 Small metacentric : 19-20 Small Acrocentric : 21-22 + Y
Nomenclature
High and Low resolution
Karyotype designation
• Suspected known chromosomal syndrome • Disorders of sexual development • Short stature in a prepubertal female • Unexplained Intellectual disability especially when associated with • Multiple organ malformations • Facial dysmorphism • Growth retardation Indications
• Infertility • Recurrent pregnancy losses • Parents of a child with structural chromosomal abnormality • For prenatal diagnosis if one of the parent is carrier of balanced translocation • Malignancies • Special Chromosomal studies – chromosomal breakage syndrome Indications
• Low resolution limited to 5-10Mb • Labour intensive • Requirement of skilled operator • Takes 4-7 days for cells to culture • Cheaper than most other tests • Easy accessibility • Can detect balanced translocations, Inversions, Mosaicism Advantages Limitations Karyotype
• Array CGH is able to detect copy number changes at a level of 5–10 kb DNA. • It is faster and more sensitive than conventional metaphase analysis for the identification of constitutional rearrangements. • Microarray chip – A high density miniaturized array of oligonucleotides spotted onto a glass slide. Identify cryptic Deletions and Duplications. Array CGH (Microarray-based comparative genomic hybridization)
Step 1-3: patient and control DNA are labelled with fluorescent dyes and applied to the microarray which has human genome Step 4: patient and control compete to attach or hybridize to the microarray Step 5: the microarray scanner measures the fluorescent signals ( red, green, yellow) Step 6: computer analyses the data and generate a plot Technique of microarray
• The first-line test in the investigation of patients with severe developmental delay learning difficulties congenital abnormalities prenatal testing of fetus when abnormalities are detected by ultrasound scanning. Suspicion of a chromosomal disorder based on maternal serum markers levels Indications
Limitations • High cost • Cannot detect • Balanced translocations • Inversions • Del/dup across all chromosomes • Regions of LOH- s/o AR disorders • UPD- long stretch of LOH • Yield is better than traditional chromosomal analysis/ karyotyping • May detect mosaicism Advantages Array CGH
• FISH is a method that can be used to detect small deletions and duplications that are not visible using microscope analysis. • It can also be used to detect how many chromosomes of a certain type are present in each cell and to confirm rearrangements that are suspected after microscope analysis. FISH (Fluorescence in situ hybridization)
(a) The basic elements of FISH are a DNA probe and a target sequence. (b) Before hybridization the DNA probe is fluorescently labelled via the incorporation of a fluorophore (c) The labeled probe and the target DNA are denatured. (d) Combining the denatured probe and target allows the annealing of complementary DNA sequences. (e) Finally, the signals are evaluated by fluorescence microscopy Technique
(a) Chromosomal Aneuploidies (b) Chromosomal Deletion – 4p-, 5p- (c) Chromosomal microdeletion syndromes – 22q11Del, 7q11Del, 15q11Del (d) Translocation Indications
Advantages Very rapid- 3 -4 days Efficiency of Hybridization and deletion is high. Sensitivity and specificity is high. Better resolution Restricted to those abnormalities that can be detected with currently available probe. Only one or a few abnormalities can be assessed simultaneously. Has higher sensitivity for trisomy but less sensitive for detecting chromosome loss or deletion. Requires fluorescence Microscopy and an image analysis system. Limitations FISH
• PCR-based quantitation technique. • Multiplex reaction • 45 Chromosomal segment can be identified in a single reaction • Probes are amplified • And amplification depends upon specific hybridisation and ligation Multiplex ligation dependent probe amplification (MLPA)
Basic steps in MLPA 1. Denaturation 2. Hybridization 3. Ligation 4. Amplification
1. The MLPA probemix is added to denatured genomic DNA 2. The two parts of each probe hybridise to adjacent target sequences 3. Only perfectly matched probes will be ligated 20- 40 nucleotides 19-370 nucleotides
Separation and quantification by capillary electrophoresis Each peak is the amplification product of a specific probe Samples are compared to a control sample A difference in relative peak height or peak area indicates a copy number change of the probe target sequence
Results • Are always compared with a control sample • Allele copy numbers • A ratio of about 1 is obtained if both alleles are present, a ratio of about 0.5 when one allele is absent and a ratio of about 1.5 if one allele is duplicated
22q11 deletion
1. Copy Number changes (a) P245 kit - Chromosomal microdel/dup syndromes (b) P036, 070 kits – subtelomeric probes (Intellectual disability) (c) P060, 021 kits - SMA – deletion of exons (d) P034, 035 kits – DMD - deletion of exons 2. Methylation defects (a) ME028 – PWS/AS (b) ME030 – BWS/RSS 3. Known point mutations/ SNVs Indications
Advantages Cost effective Multiple areas of genome simultaneously 45 patient samples in one reaction  Rapid results Efficiency of Hybridization and deletion is high. Identify abberations that are too small to be picked up by FISH Versatile – can be customised  Only known mutations can be detected  Restricted to those abnormalities that have currently available probes.  Requires fluorescence Microscopy and an image analysis system.  Not detect mosaicism, tri/tetraploidies.  Sometimes needs confirmation by alternate method. Limitations MLPA
CLINICAL SCENARIOS
PATIENT 1 • Development delay • Examination – mongoloid slant of eyes, flat facies, small ears, b/l simian crease and clinodactyly • AVCD • What genetic tests can be done- Trisomy 21? • Conventional Karyotyping • Fluorescence in situ hybridization (FISH)
PATIENT 2 • 2 month old baby boy Referred with history of recurrent seizures Smooth perinatal transition at birth • On exam – retrognathia, cleft palate Cardiovascular system - systolic murmur at left upper sternal edge. • Investigations - Hypocalcaemia was present. Chest radiography - narrow superior mediastinum. Ultrasonography - absent thymus in the anterior mediastinum Echocardiography - Osteum secondum atrial septal defect (ASD) Absolute lymphocyte count was normal, but lymphocytes subsets testing revealed diminished activity in CD3 T cell lineage.
• Di George Syndrome Will Karyotype detect a microdeletion syndrome?? Investigation of choice – • FISH • MLPA
22q11 deletion
PATIENT 3 • Dysmorphic facial features described as elfin or pixie-like • Supravalvular aortic stenosis • Mild to moderate developmental delay with a characteristic friendly, social personality
What genetic tests can be done - deletion at 7q.11.23? • Fluorescence in situ hybridization (FISH) • MLPA with probes specific for the elastin gene, one of approximately 26 to 28 genes located at 7q11.23. • Chromosomal microarray analysis (comparative genomic hybridization) – can detect. However, above 2 tests are preferred.
PATIENT 4 • 28 year old woman with 4 Recurrent spontaneous first trimester abortions • POC – CMA was done – Trisomy 13 • Karyotype of couple was done to check if any of them is balanced translocation carrier • Karyotype of husband- 46XY t(4,13)
• Chromosomal Aberration • Down Prader Willi Unknown Chromosomal syn • Karyotype FISH CMA • FISH MLPA
Cost Karyotype FISH MLPA CMA Cost PGIMER - 250 1500-2500 INR PGIMER – 1200 5-8000 INR PGIMER - 2500 8000 PGIMER - soon 12-15000 TAT 7- 10 days 72 hours 7-14 days 2 weeks
• Gene – fundamental unit of heredity. • A sequence of DNA involved in producing polypeptide chain. • Includes coding segments (Exons) and intervening sequences (introns). • It is functionally defined by its product. Genes
Is a Pathogenic Variation in DNA – Point mutation - structural change • Deletion • Insertion • Substitution - replacement of single base nucleotide with another • Frameshift – generally leads to non sense and chain termination Functional change - can lead to missense or nonsense change. Mutation
Disorders of the Gene When to suspect Rare Mendelian disorders? / How to identify? What are types of Mendelian Diseases? What are the tests available for testing disorders of gene? Indications Technique Advantages Limitations
Inheritance Pattern- • Family Pedigree and Family history • Consanguinity • Similar history in Parents • Siblings • Maternal uncle When to suspect a Mendelian disorder? / How to identify? Children • Constellation of clinical features consistent with known disorder eg. – Achondropalsia, IEM, Hemophilia, Marfan syndrome • Facial Dysmorphism consistent with a mendelian disorder eg.- Kabuki syndrome, Rubinstein taybi.. • Dysmorphism with or without behavioral problems • Unexplained GDD and ID with normal chromosomes
• Autosomal Dominant • Autosomal Recessive • X linked Dominant • X linked Recessive What are types of Mendelian Diseases?
What are the tests available for disorders of Gene? Targeted • PCR • Sanger Sequencing - First generation Non Targeted • Next Generation Sequencing • Next generation Sequencing NGS Panels Clinical Exome sequencing Whole Exome sequencing Whole Genome sequencing • PRE and POST test counselling is of utmost importance.
Single Gene • Sanger sequencing (hotspot mutation) Multiple Genes • Sequencing Panel • Clinical exome Non Targeted • Perform Whole Exome Seq (WES) Detailed clinical and family history Identify Phenotype with or without family history Couldn’t identify known syndrome No outcome from WES Non Targeted • Perform Whole Genome Seq
• DNA sequencing is the process of determining the sequence of nucleotide bases (eg: ATCGAT) in a piece of DNA • Frederick Sanger - two times Nobel Prize winner • Human Genome Project – 1990-2003 – determining base pairs that make up human DNA – 2.7 billion USD Sanger Sequencing
• Principle – is a DNA sequencing method in which target DNA is denatured annealed to oligonucleotide primers (fluorescently labelled) extended by DNA polymerase using mixture of dNTPs and chain terminating ddNTPs • ddNTPs – lack 3’OH group to which next dNTP of growing chain is added. Hence, no more nucleotides can be added. Sanger Sequencing
• For confirmation of a variant found by NGS • Once a variant is detected in an affected patient by NGS, Sanger sequencing is performed For • Segregation analysis in parents • Family Screening • For prenatal testing, in a future sibling of an affected child • A small gene - cost effective to perform Sanger Sequencing rather than NGS • For common hotspot mutations in certain diseases like – Cystic Fibrosis, Achondroplasia Indications
Limitations • Targeted • Can only sequence short pieces of DNA--about 300 to 1000 base pairs. • High cost • Laborious • Great Accuracy • Gold standard for SNV • Cost depends on the region/ length of DNA/ no. of exons. Advantages Sanger Sequencing
• Sequencing all of the protein-coding regions of genes in a genome (known as the exome). • Exome is only 1% of total genome Whole Exome Sequencing
•To detect Single Nucleotide Variation (SNV) For Mendelian disorders Polygenic Disorders – such as Alzheimer's disease Cancer Syndromes Indications
Advantages • Non focused • All 30,000 genes • Once data generated, can be stored, reanalyzed in future. • Large amount of data • Results need clinical correlation • Parents and sibling testing is required in most cases • Many times Variants of unknown significance is found – needs further testing to classify it into benign or pathogenic • Test is costly – price around 20,000 INR • Time taken for results – 3- 6 weeks • Ethical issues - newborns, adult onset disorders, predictive testing – No strict guidelines in India Limitations Whole Exome Sequencing
• 99% of the human genome is not covered by exome sequencing. • Sequences all coding as well as non coding regions (Exons + Introns) • Presently, rarely practical in the clinical context high costs time associated with sequencing full genomes. Whole Genome Sequencing
Advantages • All 30,000 genes – coding and non coding • Identification of new genes • Deep Intronic variants • Research - Modifier genes/ novel findings • Large amount of data – difficult interpretation • Parents and other sibling testing is required in most cases • Many times Variants of unknown significance is found – needs further testing to classify it into benign or pathogenic • Test is costly – price around 70,000 INR • Time taken for results –6 weeks Limitations Whole Genome Sequencing
Cost Sanger Seq NGS panel Clinical exome Whole exome Whole genome Cost PGIMER – 1250 per exon ~3500 per exon PGIMER – 6000 - ~15,000 - ~20,000 - ~60-80k TAT ~14 days ~3 weeks ~3 weeks ~ 4 weeks ~6 weeks
ABCD1 Adrenoleukodystrophy AGL Glycogen storage disease IIIa, IIIb ARSA Metachromatic leukodystrophy ARSB Mucopolysaccharidosis type VI (Maroteaux-Lamy) ASPA Canavan disease CFTR Congenital bilateral absence of vas deferens, Cystic fibrosis, Sweat chloride elevation without CF, {Pancreatitis, hereditary} G6PC Glycogen storage disease Ia GAA Glycogen storage disease II GALC Krabbe disease GALNS Mucopolysaccharidosis IVA GBA Gaucher disease GCDH Glutaricaciduria, type I GLB1 GM1-gangliosidosis, type I, II, III, Mucopolysaccharidosis type IVB (Morquio) GNPTAB Mucolipidosis II & III alpha/beta NGS Panel
HEXB Sandhoff disease, infantile, juvenile, and adult forms IDS Mucopolysaccharidosis II Hunter Syndrome MMAB Methylmalonic aciduria, vitamin B12-responsive, due to defect in synthesis of adenosylcobalamin, cblB complementation type MMADHC Homocystinuria, cblD type, variant 1; Methylmalonic aciduria and homocystinuria, cblD type; Methylmalonic aciduria, cblD type, variant 2 MUT Methylmalonic aciduria,†mut(0) type NPC1 Niemann-Pick disease, type C1, D NPC2 Niemann-pick disease, type C2 PCCA Propionicacidemia PCCB Propionicacidemia SGSH Mucopolysaccharidosis type IIIA (Sanfilippo A) SMPD1 Niemann-Pick disease, type A, B IDUA Mucopolysaccharidosis Ih MMAA Methylmalonic aciduria, vitamin B12-responsive IVD Isovaleric acidemia GLA Fabry disease HEXA Tay-Sachs disease; GM2-gangliosidosis, several forms
•Gene • Point Mutation – Thal, CF Exonic Del/ Dup Syndrome not identified • PCR • Sanger Seq MLPA NGS Syndrome Identified – has multiple genes Or single large gene with no hotspot
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