Page 1338 - Clinical Immunology_ Principles and Practice ( PDFDrive )

CHaPTEr 96 Molecular Methods 1299

to hybridize region-specific DNA probes directly to a chromosome sequences and misaligned intermediates during DNA synthesis.
spread. The technique, called fluorescence in situ hybridization An entirely different mechanism for mutation is expansion of
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(FISH), allows the detection of signals from the chromosome short repeat segments (most often trinucleotides). So far, no
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spread to directly localize genes. FISH is used for cytogenetic disorder that affects the immune system has been associated
examination of tumors, leukemias, and lymphomas but now with this mechanism.
has been largely replaced by array comparative genome hybridiza- Heritable mutations can arise in either the male or the female
tion (CGH), which examines many hundreds of thousands of germline, but the gender-related frequency is influenced by the
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positions using oligonucleotide probes. High-density oligonucle- mutational mechanism. Chromosomal nondisjunction, for
otide arrays allow for both standardization of reagents between example, occurs predominantly during female meiosis and has
diagnostic laboratories and extensive customization for particular a strong maternal age effect. 14,15 The fact that spermatogonial
disease applications. These methods are detailed below. Pathogenic proliferation occurs throughout life and involves many more cell
copy number variants (CNVs) are a major cause of birth defects replications than oogenesis increases the single base mutation
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and intellectual disability, so evaluation for CNVs should be frequency in the paternal germline. 16,17 Clinicians must cope with
considered a first-line test for any infant or child with a complex both locus and allele heterogeneity. Locus heterogeneity means
presentation of immune deficiency. In DiGeorge syndrome (DGS), that the same or a similar phenotype results from mutation in
the characteristic deletion of chromosome 22q11 can be detected one of several different genes. For example, severe combined
by microarray. FISH should not be used because microarray not immunodeficiency (SCID) can result from mutation in the adenos-
only provides high-resolution determination of the deletion size ine deaminase, interleukin-2 (IL-2) receptor γ-chain genes, JAK3,
but also leaves open the possibility of detecting other unsuspected and so on. Allelic heterogeneity means that the disease is caused
DNA copy number abnormalities. by different mutations in the same gene. In X-linked disorders,
allelic heterogeneity is typically high because affected individuals
Mutation and Pathogenic Variants have reduced reproduction (negative evolutionary selection), and
KEY CONCEPTS most mutations are lost in the population after a few generations.
At autosomal loci some mutations have reached appreciable
Mutation frequency as a result of demographic processes (e.g., founder effect).
In the heterozygous state, recessive mutations can be weakly
• Mutation can occur by deletion, insertion, or duplication of short or
long DNA segments. deleterious, neutral, or actually confer a small advantage. Because
• Single-base mutations can be caused by replication errors or by chemical the mutant alleles are common, it is possible to test affected
deamination of methylcytosine. individuals and potential carriers directly for those mutations.
• Single-base mutations can affect protein coding sequence, regulatory This is the basis for population screening for carrier status in
sequences, or the RNA splice signals within a gene. cystic fibrosis. However, none of the immune deficiencies results
• The parental origin of mutations (maternal or paternal) affects those from common mutations that would permit efficient screening.
mutational mechanisms most likely responsible.
• A disease can be caused by mutations in several alternative genes In the autosomal recessive immune deficiencies, most patients
(locus heterogeneity). have two different, very rare mutations (compound heterozygosity)
• Most primary immunodeficiencies occur because of different kinds at the disease locus. If there is known parental consanguinity or
of mutations in particular genes (allelic heterogeneity). the family comes from an isolated population, the affected
individual may be homozygous for a rare mutation.

DNA variants can involve single nucleotide substitutions, small X-Chromosome Inactivation
or large deletions, insertions, inversions, duplications, or repeat Disturbances in the pattern of X inactivation are interesting
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expansions. Some variants have no measurable or functionally phenomena in women who are carriers of X-linked disorders,
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significant phenotypic effect. They become one of the huge pool including several immune deficiencies. Measurement of
of neutral polymorphisms in the genome. Single-base substitu- X-chromosome activity in the blood cells of normal women
tions in a triplet amino acid codon often disturb the normal shows that, on average, the contribution of maternal and paternal
function of proteins. These are described as missense (i.e., causing X chromosomes is approximately 50 : 50. Extreme skewing of X
an amino acid substitution) or nonsense (i.e., terminating transla- inactivation often results from abnormal proliferation of blood
tion) mutations. Mutations affecting regulatory or splice signal cells. X-inactivation analysis can be used to demonstrate clonal
sequences in RNA can also be deleterious. The primary mecha- growth of premalignant (e.g., myelodysplasia) and malignant
nisms of mutation are misincorporation of nucleotides and faulty cells. In a female carrying mutations at the X-linked severe
repair of chemically damaged nucleotides (e.g., 8-oxodG) during combined immune deficiency (XSCID; OMIM 300400), agam-
DNA replication by DNA polymerases. Another important maglobulinemia (XLA; OMIM 300300), or Wiskott-Aldrich
mechanism underlying single base or “point” mutations occurs syndrome (WAS; OMIM 301000) loci, cell competition and
at CpG dinucleotides. The cytosine is often methylated, and compensation mechanisms lead to a reduced contribution of
chemical deamination of the methylated cytosine gives the base cells expressing the mutant allele among affected cells. In XLA,
thymidine. At the next round of replication the CG may be the B-cell lineage shows selective use of the nonmutant active
changed to TG or CA, depending on which DNA strand was X chromosome. In XSCID, skewing of X inactivation is observed
altered. Small insertion and deletion (indel) mutations are also in B cells, T cells, and natural killer (NK) cells. In WAS, some
very common. Indels occurring in protein coding sequence are degree of skewing of X inactivation results from defective
usually deleterious, as they can cause frameshifts (changing the hematopoietic stem cell (HSC) activity. Historically, skewed X
reading frame, leading to translation termination within a few inactivation was very helpful in linkage mapping of these condi-
codons) and account for almost 25% of known human disease tions. As a clinical test, X inactivation now has reduced importance
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causing mutations. They result from strand slippage in repetitive compared with direct mutation analyses.