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Chapter 2 : Molecular Methods for Diagnosis of Genetic Diseases Involving the Immune System

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Molecular Methods for Diagnosis of Genetic Diseases Involving the Immune System, Page 1 of 2

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Abstract:

Disorders of the immune system affect a significant number of individuals, with prevalence estimates (per 100,000) determined by registries ranging from 5.6 in Australia (1) to 4.979 in France, 2.6 in The Netherlands, and down to 1.377 in Germany (2). Accurate diagnosis of immune system disorders may allow early intervention prior to extensive illness for severe disease, or more specific treatment in the case of later-diagnosed or milder disease. This chapter explores some of the current molecular methodologies for detecting disorders of the immune system and highlights specific pitfalls that may hinder accurate diagnosis.

Citation: Hsu A. 2016. Molecular Methods for Diagnosis of Genetic Diseases Involving the Immune System, p 5-18. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch2
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Figures

Image of FIGURE 1
FIGURE 1

Sanger sequence chromatograms. (A) Normal sequence displaying a single base call at each location. The top row demonstrates sequence from early in the chromatogram, ~150 bp into the sequence, while the middle row is the same location but near the end of the chromatogram, at ~750 bp. The bottom chromatogram shows readable sequence but significant nonspecific baseline noise, making interpretation less than ideal. (B) An example of wild-type sequence compared to a heterozygous mixed base. The wild-type blue (C) peak has decreased in height, and the red (T) allele overlays the same location. (C) An example of wild-type sequence (top tracing) compared to 4-bp duplication (bottom tracing). The bases listed below the tracing demonstrate how to determine the variation causing the heterozygous peaks. Bases in black are wild type, while bases in red signify the mutant allele. Boxed bases correspond between wild type and mutant, allowing identification of the inserted bases indicated by a bracket.

Citation: Hsu A. 2016. Molecular Methods for Diagnosis of Genetic Diseases Involving the Immune System, p 5-18. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch2
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Image of FIGURE 2
FIGURE 2

Real-time qPCR plot. (Left) Amplification plot of duplicate dilutions of a known standard TREC plasmid. The axis is PCR cycle number; the axis is ∆Rn, which is Rn (the ratio of the reporter signal normalized to a constant fluorescent signal) minus the value of Rn at baseline; the green line is threshold value. Threshold cycle (C) is defined as the cycle number at which the amplification plot crosses the designated ∆Rn threshold (blue dots). (Right) Logarithmic plot of the same standards as well as two duplicate unknown samples, and , for which the C value, and therefore copy number or concentration, can be derived by comparison with the standard curve. (Figure courtesy of Jennifer M. Puck.)

Citation: Hsu A. 2016. Molecular Methods for Diagnosis of Genetic Diseases Involving the Immune System, p 5-18. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch2
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Image of FIGURE 3
FIGURE 3

High-resolution comparative genomic hybridization array spanning 375,000 bases of the region surrounding on the X chromosome. Plots are log 2 of the intensity of the patient sample compared to all samples run on the array. Panel A demonstrates one female patient with a large deletion encompassing , , , and as evidenced by the contiguous −0.5 intensity signal, which then resolves to 0, indicating the presence of two alleles.

Citation: Hsu A. 2016. Molecular Methods for Diagnosis of Genetic Diseases Involving the Immune System, p 5-18. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch2
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Image of FIGURE 4
FIGURE 4

Conserved splicing motifs. Gray boxes indicate exons; solid lines are introns. 3′ss begins with the branch point located between 15 and 40 bases 5′ to the exon, noted in brackets, with the loose motif of ynyuray. Y represents pyrimidines (C or T); N is any base; U is uracil, the RNA equivalent of T, R is A or G. Following the branch point sequence is the polypyrimidine tract, Y(n), where n ranges from 5 to 20 bases of mostly pyrimidines, followed by the 5′ss invariant AG (bold) as the last two bases. The first base of the exon is usually a G. At the end of the exon, the final base is again usually a G, followed by the invariant GT (bold) at the start of the intron. The 5′ss motif is GTRNG.

Citation: Hsu A. 2016. Molecular Methods for Diagnosis of Genetic Diseases Involving the Immune System, p 5-18. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch2
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Image of FIGURE 5
FIGURE 5

Analysis of NEMO variants. (A) Intracellular flow cytometry for NEMO protein. The dotted line is isotype control; the solid line is specific NEMO staining. The top panel is the normal control, showing an FI of 30.6. The middle panel is patient 1, with all cells showing specific staining but with a reduced intensity and an FI of 12.6. The bottom histogram is patient 2, again demonstrating that all cells contain NEMO protein, but less than both the control and patient 1, with an FI of 5.4. (B) The top panel shows the genomic organization of , the gene encoding NEMO. Gray boxes are exons, narrow boxes are untranslated, and small white box insets are uORFs. Locations of patient genomic mutations are noted, with wild-type sequence above the genomic structure, mutant motif below, and the single base mutation in bold. The lower panel depicts the structure of the cDNA, showing aberrant splicing. Patient 1 has a 153-bp deletion of exon 5, noted by the thin line in the middle of the cDNA. Patient 2 has a deletion of a portion of the 5′ untranslated region, including one of the two uORFs.

Citation: Hsu A. 2016. Molecular Methods for Diagnosis of Genetic Diseases Involving the Immune System, p 5-18. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch2
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Image of FIGURE 6
FIGURE 6

(A) Sanger sequence chromatograms from a patient demonstrating mosaicism for a mutant allele. The left column shows coding sequence; the right column, the intronic SNP 132 bp 3′ to the mutation but contained within the same PCR product. The top row is from a healthy control, demonstrating the wild-type G at c.1145 and homozygous G at the intronic SNP. The middle row is from the affected son, with heterozygous peaks at the site of the mutation, c.1145G>A, and homozygous G at the SNP. The bottom row is from the father, showing a reduced peak height of the wild-type G allele (black) compared with the healthy control and the presence of a small green peak corresponding to the mutant allele (arrow) but at less than heterozygous levels. This is in contrast to his intronic SNP, which demonstrates heterozygosity (arrow). (B) Sequence tracings of the X-linked gene. The top tracing is from a healthy control, showing only wild-type sequence. The middle tracing is from the patient's mother, who is heterozygous for the c.260C>T mutation, showing the reduced T peak (red) and the presence of the mutant C peak (blue). The bottom tracing, from a male patient with the inherited mutant C allele from his mother, shows a small, wild-type T peak (red) underneath (arrow).

Citation: Hsu A. 2016. Molecular Methods for Diagnosis of Genetic Diseases Involving the Immune System, p 5-18. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch2
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Image of FIGURE 7
FIGURE 7

genomic structure showing exons (black boxes) and introns (black line). Below is intronic sequence from three patients, demonstrating the intronic point mutation (bold) in each that creates a cryptic 5′ss allowing for inclusion of a cryptic exon (white boxes). Wild-type sequence for each site is shown below the patients’ mutant sequences. The insertion of the cryptic exon results in a frameshift for the first two patients, while the third, with an insertion of 120 bp, is predicted to have a 40-amino-acid insertion.

Citation: Hsu A. 2016. Molecular Methods for Diagnosis of Genetic Diseases Involving the Immune System, p 5-18. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch2
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Image of FIGURE 8
FIGURE 8

Reduced allelic expression in MonoMAC syndrome. (A) The top row shows genomic sequence from two patients demonstrating heterozygosity for a SNP within the cDNA transcript of . The bottom row shows cDNA sequence from full-length RT-PCR. In patient 1, only the T allele is present (arrow), indicating loss of the transcript containing the C allele, while patient 2's sequence contains the C allele with only a small peak of the G allele (arrow). These sequences demonstrate uniallelic expression leading to haploinsufficiency of . (B) At the top is genomic sequence from two affected sisters; the proband is heterozygous for three known SNPs found within the cDNA, while her sister is homozygous, allowing determination of the shared mutant allele haplotype (boxed bases). At the bottom is cDNA sequence from the proband's CD3 and CD3 PBMCs, revealing decreased levels of the mutant allele as represented by peak height (arrows). (C) genomic locus showing three reported isoform structures. The dashed box indicates a conserved intron 5 region with a high GERP score, DNase I hypersensitivity, and strong transcription factor binding. Zoom of bases highlights the E-box/GATA composite element (boxed); the 28-base deletion seen in the first patient (underlined sequence); and the ETS motif with a C>T mutation seen in five patients (asterisk), including the sisters shown in panel B.

Citation: Hsu A. 2016. Molecular Methods for Diagnosis of Genetic Diseases Involving the Immune System, p 5-18. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch2
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Image of FIGURE 9
FIGURE 9

Framework for molecular and genetic diagnosis in immunocompromised patients, beginning with defining of patient phenotype, proceeding to performing genomic analysis, and concluding with inferred or demonstrated pathogenicity of mutation.

Citation: Hsu A. 2016. Molecular Methods for Diagnosis of Genetic Diseases Involving the Immune System, p 5-18. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch2
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Tables

Generic image for table
TABLE 1

Comparison of three major NGS platforms

Citation: Hsu A. 2016. Molecular Methods for Diagnosis of Genetic Diseases Involving the Immune System, p 5-18. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch2
Generic image for table
TABLE 2

Online resources for variation analysis

Citation: Hsu A. 2016. Molecular Methods for Diagnosis of Genetic Diseases Involving the Immune System, p 5-18. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch2

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