MTPC 140: Molecular Biology and Diagnostics

Compiled by: Maverick V. Sustiguer, RMT

©2021
MOLECULAR DIAGNOSIS OF GENETIC DISEASES
©2021
One of the defining achievements of the early 21st century is the sequencing and alignment of more than 90% of the
human genome. Of course, there is not a single human genome: individuals differ from each other by about 0.1% and
from other primates by about 1%. Variation comes in many different forms, including single base changes and copy
number changes in large segments of DNA. Even more challenging than sequencing the whole genome is documenting
and understanding the clinical significance of human sequence variation. We are still very early in our understanding of
the human genome.
GENOMIC TERMS AND DEFINITIONS
Annotation: Biologic information attached to genomic sequence.
Annotation Track: Optional metadata in a genome browser that allows viewing of genes, exons, SNVs, repeats, etc.
Anticipation refers to a progressive increase in severity and/or decrease in age of onset of a genetic disorder in
subsequent generations of a family.
Assembly: Reconstruction of short sequence reads on a scaffold of reference DNA.
Binary alignment nap (BAM): After alignment to a reference genome, the aligned data for each read produces a
sequence alignment map (SAM file). The BAM file is the binary equivalent of the SAM file and allows for
efficient random access of the data.
Browser extensible data (BED): A tab delimited text file that defines the data lines in an annotation track, including the
chromosome name, the starting and the ending positions.
Contig: A linear stretch of consensus sequence assembled from smaller overlapping sequence fragments.
Copy number polymorphism (CNP): A copy number variant present at more than 1% in a population.
Copy number variant (CNV): A structural variant of a large region of the genome that has been deleted or duplicated.
De novo assembly: Formation of a contig without using a reference sequence.
Deletion: A DNA sequence that is missing in one sample compared to another. Deletions may be as small as one
nucleotide or as large as an entire chromosome.
FASTA File: A nucleotide sequence text file.
FASTQ file: A text output file of sequencing reads in a run, along with the quality scores of each position.
Fusion: A translocation, inversion, large deletion, or large duplication resulting in a hybrid gene formed from originally
separate genes.
Heterogeneity: The number of different genes, or the variety of mutations within a single gene, that can cause the same
disease.
Heteroplasmy: A mixture of more than one type of mitochondrial sequence in one cell.
Imprinting: The differential expression of a gene in an offspring, depending on whether it was inherited from the mother
or the father, or sometimes on other epigenetic influences.
Indel: Originally referred to a unique class of sequence variants that included both an insertion and a deletion resulting in
an overall change in the number of base pairs. Today more commonly refers to either insertions or deletions or a
combination thereof.
Insertion: An extra DNA sequence that is present in one sample compared with a reference sequence.
Intergenic: DNA sequence between genes.
Missense: A nucleotide substitution that changes a codon to the code for a different amino acid. Although these sequence
changes are commonly referred to as missense “mutations,” this is strictly a misnomer because missense variants
may be benign and cause no disease.
Mutation: A disease-causing sequence variation. Historically, the term has been interchangeable with variant to describe
any change in DNA sequence regardless of relation to disease causation. For current clinical descriptions or
reporting, the use of mutation is reserved for the scenario when disease causation is known.
Nonsense: A nucleotide substitution that results in a stop codon, prematurely terminating the protein.
Nonsynonymous: Nucleotide substitutions that are predicted to change the amino acid coding. These substitutions
include both missense and nonsense substitutions.
Oligonucleotide: A short single-stranded polymer of nucleic acid.
Penetrance: The proportion of individuals who, having inherited a mutant disease gene, will actually display the disease
phenotype.
Phred score: Estimate of the error probability for a base called in DNA sequencing. It is represented as a Q-score; the
higher the number, the higher the probability of a correct call.

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 MTPC 140: Molecular Biology and Diagnostics
Compiled by: Maverick V. Sustiguer, RMT
©2021
Plasmid: An extrachromosomal ring of double-stranded, closed DNA found in bacteria.
Pseudogene: A genetic element that does not code for a functional gene product, usually because of accumulated ©2021
sequence variations.
Sequence alignment map (SAM file): A file generated by alignment of sequence data to a reference genome. This file
type is often converted to a BAM file to save space.
Short tandem repeat (STR): A simple sequence repeat that is 1-13 bases long.
Simple sequence repeat (SSR): A sequence from 1-500 bases that is repeated end to end. If the repeat unit is 1-13 bases,
it is a microsatellite or STR. If the repeat is 14-500 bases it is a minisatellite.
Single nucleotide polymorphism (SNP): A benign single nucleotide variant (substitution, deletion, or insertion) that
occurs in a population at a frequency of at least 1%.
Single nucleotide variation (SNV): A single nucleotide variant (substitution, deletion, or insertion). SNVs may be
benign or may cause disease.
Structural variation: A region of DNA greater than 1000 bases in size that is inverted, translocated, inserted, or deleted.
Synonymous variant: A nucleotide change that results in no change to the predicted amino acid sequence. Although
synonymous variants are typically considered to be benign since there is no protein coding change, there is the
possibility of pathogenicity by changes in splicing, gene expression or mRNA stability.
Transposon: A mobile genetic element that can delete and insert itself variably into the genome.
Variable expressivity: The appearance of different signs and symptoms of a disorder in individuals inheriting the same
mutation(s).
Variant call format (VCF): After aligning all reads onto a reference sequence, variants that are different from the
reference genome at a given nucleotide position are stored in a text file in a specific format.
Variation: A change in DNA sequence. It may be benign or may cause disease.
HUMAN GENOMIC DATABASES
Comprehensive
 NCBI (National Center for Biotechnology): ncbi.nlm.nih.gov/genome
 Ensembl: ensembl.org/index.html
 UCSC (University of California Santa Cruz): genome.ucsc.edu/
Genes and Disease
 OMIM (Online Mendelian Inheritance in Man): ncbi.nlm.nih.gov/omim
 ClinVar: ncbi.nlm.nih.gov/clinvar/
 Decipher: decipher.sanger.ac.uk/index
Sequence Databanks
 NCBI GenBank: ncbi.nlm.nih.gov/Genbank/
 EMBL (European Molecular Biology Laboratory)-Bank:www.ebi.ac.uk/embl
 DDBJ (DNA Data Bank of Japan): ddbj.nig.ac.jp/
MicroRNAs
 miRBase: mirbase.org
General Variation Databases
 Leiden Open Variation Database: lovd.nl/3.0/home
 Human Genome Mutation Database: www.hgmd.cf.ac.uk/ac/index.php
 Short Genetic Variations (dbSNP): ncbi.nlm.nih.gov/projects/SNP/
 1000 Genomes: www.1000genomes.org/
 Exomes (ExAC): exac.broadinstitute.org/
Specialized Variant Databases
 Database of Genomic Structural Variation (dbVar): ncbi.nlm.nih.gov/dbvar
 Database of Genomic Variants (DGV): dgv.tcag.ca/dgv/app/home
 Retrotransposons: dbrip.brocku.ca/
 Haplotypes (HapMap): hapmap.org/
Nomenclature
 HUGO (Human Genome Organization) Gene names:genenames.org
 HGVS (Human Genome Variation Society) Sequence variants: www.hgvs.org/mutnomen

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 MTPC 140: Molecular Biology and Diagnostics
Compiled by: Maverick V. Sustiguer, RMT
©2021
CHOICE OF TECHNIQUES
©2021
 PCR
 Southern Blotting
 Allele-specific probe hybridization
 qPCR
 Nucleic acid Microarrays
 Invader Assay
 Amplification Refractory Mutation System (ARMS)
 Oligonucleotide Ligation Assay (OSA)
 Multiplex Ligation-dependent Probe Amplification (MLPA)
 Denaturing Gradient Gel Electrophoresis (DGGE)
 Mass Spectrometry
 Next-generation Sequencing (NGS)

In choosing a technique, you must consider the following factors:

1. The present knowledge of the gene(s) associated with the disease in question.
 Direct gene/mutation analysis can be done if the causative agent has been identified.
 Linkage analysis using polymorphic DNA markers nearby on the same chromosomes can be employed if
the causative gene has not been identified.
2. The degree of molecular heterogeneity.
The greater the heterogeneity, the more difficult, labor-intensive, and expensive the DNA test becomes.

To some extent, the choice of technique will also depend on the application or clinical indication. In medical genetics,
these applications fall into five major areas:
1. Carrier Screening: detection of recessive mutations in healthy individuals for purposes of genetic counseling
and family planning
2. Newborn Screening: aims to identify relatively prevalent inherited defects in otherwise asymptomatic
individuals to ascertain affected babies early in life so that treatment can be initiated before irreversible damage
occurs
3. Diagnostics Testing: performed on a symptomatic individual
4. Pre-symptomatic DNA Testing: is applied primarily to late-onset dominant disorders, in which the offspring of
an affected parent are aware that they are at 50% risk for having inherited the disease gene and desire to know
their status before its clinical onset to make informed reproductive, employment, and lifestyle decisions or to
initiate surveillance or preventive interventions
 Prenatal Testing: detection of genetic disease in the fetus
CLINICAL UTILITY OF MOLECULAR METHODS
• Targeted PCR amplification is most useful in the identification of common pathogenic point mutations.
• msPCR is capable of determining the methylation status of DNA and is often used in the diagnosis of imprinting
disorders.
• Full-gene Sanger sequencing is typically used to identify pathogenic point mutations and small insertion or deletion
mutations in disorders associated with a single disease-causing gene.
• MPS is the ideal technology to use for the identification of a pathogenic point mutation or a small insertion or deletion
mutation in disorders caused by mutations in any of a number of genes.
• MLPA is most useful in the detection of large deletions or duplications in three or fewer disease-causing genes.
• Array-based technology is most useful in the simultaneous detection of large deletions or duplications in numerous
genes.

It is assumed that the test is capable of detecting a given mutation whenever it is present; it is just that many rarer
mutations will not be targeted by the assay, so that some proportion of carriers will be “missed”—a sort of “clinical false-
negative” (Palomaki et al, 2004). It is important to keep in mind that this discussion is about clinical sensitivity, not
analytical sensitivity.

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 MTPC 140: Molecular Biology and Diagnostics
Compiled by: Maverick V. Sustiguer, RMT
©2021
The ultimate gold standard for identifying all possible mutations would of course be whole-genome sequencing. It took
13 years to completely sequence the consensus human genome under the Human Genome Project using conventional ©2021
Sanger sequencing platforms. At present, the major clinical application of this technology in genetics is for the diagnosis
of syndromic-appearing conditions for which standard single-gene and other clinical laboratory tests have been
unrevealing.
INHERITED CANCER
• Disease can result from the inheritance of a single activating mutation in a proto-oncogene.
• Disease can result from the inheritance of a single loss of function mutation in a tumor suppressor or mismatch repair
gene followed by somatic inactivation of the second allele.
• Age of onset and type of tumor(s) observed within family members are variable.
• MPS-based disease specific tumor panels are currently most often used to identify the gene mutation segregating in the
family.
• Routine clinical surveillance screening is initiated in mutation-positive asymptomatic family members.
• Genetic counseling is an important component of patient care and management.
INHERITED DISEASES
• Mitochondrial and imprinting disorders follow non-Mendelian patterns of inheritance.
• Complex diseases result from the contribution of both genetic and environmental factors and do not follow Mendelian
inheritance patterns.
• For most genes, pathogenic mutations are located throughout the gene and are heterogeneous in nature.
• For some disease genes, the type of mutation and effect on the encoded protein can predict the clinical phenotype and
identify targeted therapy for patient care.
• Diagnostic DNA testing is complicated by genetic and allelic heterogeneity.
• Genetic counseling is an important component of patient care and management.

IDENTITY ANALYSIS: USE OF DNA ANALYSIS IN PARENTAGE, FORENSIC, AND MISSING PERSONS
TESTING
• DNA has sufficient variation among individuals to discriminate all individuals who have ever lived on the earth or will
in the foreseeable future. Furthermore, DNA persists as an identification taggant over the lifetime of the individual and
can be obtained from all tissues and fluids, even trace amounts.
• Standardized marker systems with known allelic polymorphisms are used in testing panels. Alleles with short tandem
repeats form the basis of commercially available kits.
• Forensic testing requires documentation of all steps taken during collection, extraction, and testing so results can
withstand legal challenges.
• Deoxyribonucleic acid can be obtained from any sample that contains cellular material. The stability of DNA allows it
to withstand harsh environmental conditions and long postmortem intervals.
• Care must be taken to maintain the integrity of DNA evidence and to avoid contact or exposure of the evidence to any
conditions that may contaminate and/or further degrade the original stain/evidence.
• Mitochondrial DNA can be extracted from bone and teeth after hundreds and even thousands of years because it is
small and present at hundreds to thousands of copies per cell. This type of DNA is maternally inherited and can be
useful for identifying remains.
• DNA is used in identification of remains and can be used to link a suspect to a crime, to exculpate falsely accused
suspects, to recognize serial crimes, and to distinguish copycat crimes, or to aid in accident reconstruction.
• Pathology laboratories can use DNA testing to resolve specimen mix-ups such as when samples are inadvertently
switched or pathologic material floats onto a histologic or cytologic slide.
• Standards and quality assurance guidelines for identity testing laboratories have been developed by the American
Association of Blood Banks (for parentage testing), the U.S. Department of Justice Federal Bureau of Investigation’s
Working Groups, the Forensic Science Standards Board, and the International Society for Forensic Standards (for
forensic testing).

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 MTPC 140: Molecular Biology and Diagnostics
Compiled by: Maverick V. Sustiguer, RMT
©2021
Forensic testing differs from clinical laboratory testing in several ways:
1. the forensic question is usually one of identity rather than one of presence or absence of a trait or ©2021
analyte
quantification, as is done in most clinical laboratory analyses;
2. specimens received by forensic laboratories are much more diverse than the typical blood, fluid, and tissue
samples handled by clinical laboratories;
3. clinical samples are collected under controlled circumstances, while biologic evidence is exposed to the
environment, potentially resulting in degradation, modification, and inhibition;
4. forensic samples may include DNA mixtures from multiple donors, contamination, and nonhuman sources;
5. evidentiary material cannot be replenished and may be present in only trace amounts, and
6. forensic identity testing is scrutinized in a judicial environment, requiring complete accounting for chain of
custody following its collection and strict validation of procedures.

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