00019606-201012000-00006ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2010 Lippincott Williams & Wilkins, Inc.19December 2010
p 224–231Comparison of PHOX2B Testing Methods in the Diagnosis of Congenital Central Hypoventilation Syndrome and Mosaic CarriersOriginal ArticlesJennings, Lawrence J. MD, PhD*; Yu, Min MD*; Zhou, Lili MD†; Rand, Casey M. BS‡; Berry-Kravis, Elizabeth M. MD, PhD†,§; Weese-Mayer, Debra E. MD‡ Departments of *Pathology; and‡Pediatrics, Children's Memorial Hospital, Northwestern University Feinberg School of Medicine Departments of †Pediatrics§Neurological Sciences and Biochemistry, Rush University Medical Center, Chicago, ILSources of Support: PHOX2B patent fund (Mr. Rand salary support).Lawrence J. Jennings and Min Yu are equivalent first authors.Reprints: Debra E. Weese-Mayer, MD, Department of Pediatrics, Children's Memorial Hospital, Northwestern University Feinberg School of Medicine, Center for Autonomic Medicine in Pediatrics, Mailstop #165, 2300 Children's Plaza, Chicago, IL 60614 (e-mail:
[email protected]).AbstractClinical diagnostic testing for congenital central hypoventilation syndrome (
CCHS) usually involves amplification and detection by (1) targeted mutation analysis or (2) sequence analysis. Test method performance differences are more pronounced when studying difficult templates [eg, guanine-cytosine (GC)-rich regions] or samples with abnormal allele ratios (eg, mosaicism).
CCHS, an autosomal dominant disorder with identified mosaic carriers, is caused by expansion mutations of the GC-rich polyalanine-coding region of the PHOX2B gene in greater than 90% of patients (and other PHOX2B mutations in remaining patients). The combination of a GC-rich testing region and known mosaicism in
CCHS necessitates the determination of the limit of detection for diagnostic tests. This study compared the limit of detection in
CCHS-PHOX2B testing for both targeted mutation analysis and sequence analysis. Test samples included 6 differentially sized PHOX2B expansion mutations and 1 PHOX2B deletion mutation, all diluted over a range of concentrations; and 2 mosaic dyads. The limit of detection for PHOX2B expansion mutations was 1% and 20% mutant allele concentration with targeted mutation analysis and sequence analysis, respectively. These results indicate that PHOX2B testing using targeted mutation analysis is more likely to identify even low-level mosaicism for polyalanine expansion and deletion mutations. However, sequencing of PHOX2B is required to detect single base-pair mutations that cause the remaining small subset of
CCHS cases. A combination of both the tests may be required in cases in which 1 test fails to identify the disease-causing mutation. These results can help guide clinicians when choosing a
CCHS/PHOX2B clinical diagnostic testing method and interpreting results.Congenital central hypoventilation syndrome (
CCHS) is characterized by alveolar hypoventilation and autonomic dysregulation. It usually presents in the newborn period and may be life-threatening without prompt diagnosis and intervention. At present, approximately 1000 cases of
CCHS have been identified worldwide.1 Identification of heterozygous mutations of the paired-like homeobox 2B (PHOX2B) gene is a requirement for the definitive diagnosis of
CCHS.2–8 In 90% to 95% of individuals with
CCHS, the disease-causing mutation is a heterozygous in-frame expansion of a highly-conserved 20-repeat polyalanine encoding sequence (polyalanine repeat expansion mutations, PARMs), resulting in polyalanine tracts of 24 to 33 alanines (genotypes 20/24 to 20/33).3–7,9,10 The remaining 5% to 10% of
CCHS cases are caused by missense, nonsense, and frameshift mutations in the polyalanine repeat region or elsewhere in the PHOX2B coding sequence (referred to as NPARMs).3,4,6–8As
CCHS is an autosomal dominant disorder with only one mutant allele present, the corresponding mutant allele concentration is 50% if the mutation is present in every cell in the sample. The autosomal dominant condition of inheritance implies that the mutant allele was either inherited or mutated at the time of egg fertilization and first division. If the heterozygous mutation occurred later in development, somatic mosaicism, in which a mutation is carried in only a subset of cells, would occur. For example, if the mutation occurred at the 8-cell blastula stage, there would be a mutation in 1 of the 8 cells (1 of 16 alleles), and the corresponding mutant allele concentration would be 6% (equivalent to 12% somatic mosaicism). As somatic mosaicism has been identified in approximately 5% to 10% of the seemingly unaffected parents of
CCHS patients,3,6,7,11,12 detection of low-level mosaicism becomes critically important to counsel parents regarding recurrence risks in subsequent pregnancies.The majority of
CCHS-causing PHOX2B mutations occur in the second polyalanine repeat region of exon 3 which has a high guanine-cytosine (GC) content (88%). Regions high in GC content have traditionally proven more difficult to amplify using polymerase chain reaction (PCR). Accordingly, the high GC content of this PHOX2B region makes PCR amplification of the allele with a PARM expansion difficult and increases the risk for preferential amplification of the normal allele.9 As shown by Matera et al,4 the longer PARMs in
CCHS, which lead to an expansion of the highly GC-rich area, create a difficult region to amplify with PCR and an increased possibility of allele dropout (ADO) of the expanded allele in testing of these cases. Published studies have indicated high rates of ADO with subsequent reporting of false-negative results as high as 55% in testing of
CCHS patients with PARMs.6,9 Therefore, the limit of detection (LOD) for low-level mosaicism may vary depending on the size of the PARM expansion.In clinical diagnostic testing of genetic variation, care must be taken in choosing a method of detection with the specificity, sensitivity, and LOD necessary for accurate diagnosis. In
CCHS diagnostic testing, ADO of the expanded, disease-causing PHOX2B allele has proven to become increasingly problematic with the larger expansions; thus, the LOD is predicted to increase with an increased size of the expansion.4,9 Therefore, the LOD of the testing method becomes particularly critical and the method used for the detection of variation in
CCHS probands should ideally have an established low LOD. For testing of mosaicism in parents of
CCHS probands, a very low LOD is necessary, and for purposes of genetic counseling, the LOD should be known. Using
CCHS and PHOX2B mutation detection as a representative disorder with GC-rich trinucleotide repeat expansion mutations, we hypothesized that the 2 clinically available methods for the detection of PHOX2B mutations in
CCHS, (1) PCR amplification of the 20-repeat polyalanine expansion region in exon 3 followed by targeted mutation analysis and (2) PCR amplification followed by sequencing of the gene coding regions, would differ in LOD and therefore in their ability to detect expansion and deletion mutations in cases of mosaicism. Specifically, the aim of this study was to compare the LOD of mosaicism for a range of mutations of the polyalanine repeat region of PHOX2B using these 2 clinically available testing methods.MATERIALS AND METHODSSamplesBlood samples (3 to 10 mL) were obtained from individuals with
CCHS and genomic DNA was isolated using a Puregene reagent kit (Qiagen; Alameda, CA) according to the manufacturer's instructions. DNA samples from 7
CCHS patients with known heterozygous PHOX2B mutations were used for this study. DNA from 6
CCHS patients carried a PARM with 1 normal allele of 20-alanine repeats, and 1 abnormal allele with alanine repeats ranging from 25 to 33 (genotypes designated 20/25, 20/26, 20/27, 20/30, 20/31, and 20/33). The seventh patient had a heterozygous 38 base-pair frameshift deletion (designated c722del38) that included a part of the polyalanine repeat sequence. DNA samples from 3 unaffected individuals, all carrying 2 normal 20-repeat alleles (genotype designated 20/20), were included as a negative control (n=1) and as DNA diluent to create the mosaic dilutions (n=3). Two families with both a known mosaic carrier parent and a full heterozygous mutation proband were tested to validate the experimental results.The DNA concentrations of the samples and controls were quantified using a Nanodrop spectrophotometer and standardized to a concentration of 100 ng/μL. To verify the quality of DNA, a 586 base-pair segment corresponding to exon 1 of PHOX2B was amplified using real-time PCR and SYBR green detection for all the samples and controls before making dilutions, and all the samples showed comparable crosspoints indicating equivalent concentrations of amplifiable, long-sequence DNA (data not shown). The control DNA was pooled and used to dilute patient DNA to yield the following concentrations of mutant allele for each of the 7 mutated DNA samples: 50%, 40%, 30%, 20%, 10%, 5%, 2%, and 1% (equivalent to 100%, 80%, 60%, 40%, 20%, 10%, 4%, and 2% mosaicism). All participants gave their informed consent to sample donation according to the Rush University Medical Center Institutional Review Board approved protocol and the test result analysis was included in the Children's Memorial Hospital Institutional Review Board approved protocol.PHOX2B Targeted Mutation AnalysisAmplification of the region coding for the polyalanine repeat in PHOX2B exon 3 was carried out as described earlier7 with the primer pair 5′-CCAGGTCCCAATCCCAAC-3′ (forward) and 5′-GAGCCCAGCCTTGTCCAG-3′ (reverse) (methodology and primer set patented, Rush University Medical Center, Chicago, IL; revenue supports
CCHS research). The forward primer was labeled with 6-fam. The PCR reagents and condition are the same as in sequence analysis.7 For fragment analysis, the PCR products (232 base pairs for the normal 20-repeat allele) were detected using ABI 3100-Avant (Applied Biosystems, Carlsbad, CA) with Pop 4 polymer. Rox 500 size standard was used as the size marker. Data were analyzed using the Gene Mapper software (Applied Biosystems, Foster City, CA) to determine the relative concentration of the mutant allele. This methodology represents the clinically available PHOX2B targeted mutation analysis test (www.genetests.org).PHOX2B Sequence AnalysisAmplification of the entire PHOX2B exon 3 coding region, including the polyalanine repeat, for the sequencing reactions were carried out as described earlier with the primer pair 5′-ACCCTAACCGGTGCTTTTCT-3′ (forward) and 5′-ACAATAGCCTTGGGCCTACC-3′ (reverse).7,13 The PCR products were column-purified using a Microcom Ultracel YM-100 Purification Kit (Millipore Corporation, Bedford, MA) and sequenced with the “Big Dye Terminator v1.1 Cycle Sequencing” kit (Applied Biosystems, CA) in an ABI Prism 3130 xl automatic sequencer (Applied Biosystems, Carlsbad, CA) in both the directions using 5′-CTTCACCGTCTCTCCTTCC-3′ as the forward sequencing primer and 5′-TACCCGCTCGCCCACTC-3′ as the reverse sequencing primer. The data were analyzed using Sequencing Analysis 5.1.1 and Autoassembler (Applied Biosystems, Carlsbad CA). This methodology represents the clinically available PHOX2B sequencing tests (www.genetests.org).Assessment of Limits of Detection of the 2 PHOX2B Clinical Testing MethodsMultiple experienced reviewers analyzed the results of all completed testing. The LOD for targeted mutation analysis was based on the ability of these blinded reviewers to both recognize the presence of and determine the size of the mutated allele. The LOD for the sequence analysis test was based on the ability of these blinded reviewers to both recognize the presence of and determine the sequence of the mutated allele.RESULTSLOD of PHOX2B Targeted Mutation AnalysisA total of 63 dilution samples were analyzed by the PHOX2B amplification and targeted mutation analysis (all 7 mutations at 9 concentrations: 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, and 0% mutant allele (equivalent to 100%, 80%, 60%, 40%, 20%, 10%, 4%, 2%, and 0% mosaicism). The size of the mutant allele waveform peak (peak height and area within the waveform) as shown in Figure 1, left side, was proportional to the concentration of the mutant allele (confirmed in Fig. 2). Although reduced, the mutant allele peaks for all PARM expansions could be distinguished at the 1% concentration (2% mosaicism; Figs. 1, 3), as verified by increasing the amplification and comparing the mutant allele peak at 1% to background peak noise (Fig. 1, inset A). Therefore, the LOD for the expansion mutations (25 to 33 alanines; genotypes 20/25 to 20/33) and the deletion mutation (c.722del38) were determined to be at 1% mutant allele (2% mosaicism). It is likely less than 1% mutant allele (2% mosaicism), although lower concentrations were not tested (Table 1).JOURNAL/dimp/04.03/00019606-201012000-00006/table1-6/v/2021-02-17T200027Z/r/image-tiffLimit of Mutant Allele Detection for Each PHOX2B TestJOURNAL/dimp/04.03/00019606-201012000-00006/figure1-6/v/2021-02-17T200027Z/r/image-jpegComparison of the testing methods in the detection of PHOX2B mutation using the 27-repeat polyalanine expansion mutation as a representative example at decreasing concentrations of mutant allele (range: 0% to 50%). The mutant allele has 27-polyalanine repeats (genotype 20/27). The highest allele concentration (50%) corresponds to a germline, heterozygous mutation (100% mosaicism). Targeted mutation analysis (column I). Peaks represent the differently sized PHOX2B alleles with the normal 20-alanine allele on the left and the expanded allele on the right. Peak height and area are directly proportional to the relative amount of each allele. Although faint, the mutant allele peak is detectable at a 1% concentration. Inset A, Targeted mutation analysis—amplified. With amplification, the mutant allele peak can be clearly detected above the background noise at a 1% concentration. Sequencing analysis (column II). Each different color of peak indicates a different nucleotide. The lower amplitude peaks beginning at the ninth peak on each of the higher concentration examples show the sequence of the mutant allele. Decreasing concentrations correspond to lower mutant peaks. The mutant allele sequence cannot be read accurately at 10% mutant allele (20% mosaicism), although the presence of double bands at this concentration is detected. Inset B, Sequencing analysis—amplified. When amplification is increased, the double bands at 10% concentration can be seen clearly, and using the full heterozygous (50% concentration) sequence as a reference (see positions 11, 14, and 16), the mutant allele sequence can be determined. However, at the 5% concentration, the background noise produces peaks stronger than those of the mutant allele sequence (see positions 9, 11, and 14), preventing determination of the mutant allele sequence.JOURNAL/dimp/04.03/00019606-201012000-00006/figure2-6/v/2021-02-17T200027Z/r/image-jpegCorrelation of mutant allele concentration to the relative size (area within peak) of the mutant allele peak in the targeted mutation analysis compared with the size of the normal allele peak. Using 20/27 as a representative example, this figure indicates the relationship between peak size and mutant allele concentration. Peak ratio=[area of mutant allele peak]/[(area of wild type allele peak)=(area of mutant allele peak)]. Correlation of mutant allele concentration to peak size was fitted to a linear regression line (R2=0.9959).JOURNAL/dimp/04.03/00019606-201012000-00006/figure3-6/v/2021-02-17T200027Z/r/image-jpegTargeted mutation analysis of several known congenital central hypoventilation syndrome-causing PHOX2B mutations with concentrations near the limit of detection. Results of the targeted mutation analysis at mutant allele concentrations of 5%, 2%, and 1% are shown for the 33 and 25-polyalanine repeat expansion mutations (genotypes 20/33 and 20/25) and a deletion mutation within PHOX2B (c722del38). Arrows show the location of the mutant allele peak for each of these genotypes. As shown, the 1% mutant allele is detected in all samples and can be easily discerned by zooming in (as shown previously in figure 1, inset A).LOD of PHOX2B Sequence AnalysisA total of 51 dilution samples were analyzed by PHOX2B sequencing (all 7 mutations at 7 concentrations: 50%, 40%, 30%, 20%, 10%, 5%, and 0% mutant allele, and the 27 alanine expansion was also tested at 2%, and 1% mutant allele). As the LOD for PHOX2B sequencing in all PARM samples was recognized to be at 20% mutant allele (40% mosaicism), with the mutation indeterminable at the 10% and 5% mutant allele concentrations, only the 27 alanine expansion was also tested at the 2% and 1% mutant allele to ascertain detection at these lower concentrations.The height of the mutant allele peak correlated with the concentration of the mutant allele (Fig. 1). At the 20% mutant allele concentration, the sequence of the mutant allele could be readily ascertained. When increasing the amplification of the sequencing electropherogram to allow examination of the mutant allele sequence at lower mutant allele concentrations, the mutant allele sequence was difficult to distinguish from the background “noise” at the 10% mutant allele concentration, and was impossible to differentiate from the background noise at the 5% mutant allele concentration (Fig. 1, inset B). However, at the 10% mutant allele concentration, heterozygous peaks of the mutant allele could be identified and correlated to the sequence of an identified proband, if studied simultaneously. In addition, with the increasing length of the polyalanine repeat expansions, the relative intensity of the mutant allele generally decreased corresponding to the increased difficulty of amplifying a larger, GC-rich sequence (Fig. 4). Using sequencing analysis, the LOD for each of the 6 expansion mutations was determined to be the 20% mutant allele concentration (40% mosaicism; Table 1). This is the limit at which the sequence of the mutant allele could be accurately determined without knowledge of the nature of the mutation. Despite the failure of accurate sequence determination at the 10% mutant allele concentration (20% mosaicism) by the blinded reviewer, the presence of a known (eg, familial) mutation could be detected at this concentration and related to the known reference sequence (Fig. 1 inset B, Fig. 5). The sequence of the deletion mutation was more clearly detected at 10% mutant allele (20% mosaicism) as it is smaller than the normal 20-repeat sequence, and thus preferentially amplified (Table 1).JOURNAL/dimp/04.03/00019606-201012000-00006/figure4-6/v/2021-02-17T200027Z/r/image-jpegSequencing analysis of several known
CCHS-causing PHOX2B mutations with concentration near the limit of detection. The concentration of the mutant allele in each sample is 20% (40% mosaicism). As shown, the mutant sequence can be determined for all the samples.JOURNAL/dimp/04.03/00019606-201012000-00006/figure5-6/v/2021-02-17T200027Z/r/image-jpegTesting of mosaic carriers in families with congenital central hypoventilation syndrome probands. Family 1 (upper half of the figure). Proband has heterozygous 27-alanine expansion mutation. Parent is a mosaic carrier of the same mutation. Column I. Results of the targeted mutation analysis in the mosaic carrier show both the 20-alanine repeat allele peak and the 27-alanine repeat allele peak. The 27-alanine repeat allele peak shows a marked decrease in amplitude as compared with the 20-alanine repeat allele peak, indicating mosaicism [mutant allele determined to be 9% concentration (18% mosaic)]. Column II. Results of sequence analysis in 20/27 proband and mosaic carrier. A, Mutant allele sequence of the proband with full heterozygous 20/27 mutation can clearly be determined. B, Mutant allele sequence of the mosaic carrier is indeterminable. C, Even at an increased amplitude such that the tops of the peaks are no longer shown in the figure, and using the proband sequence as a reference, the mutant allele sequence of the mosaic carrier is indeterminable. Family 2 (lower half of the figure). Proband has heterozygous 26-alanine expansion mutation. Parent is a mosaic carrier of the same mutation. Column I. Results of the targeted mutation analysis in mosaic carrier show both the 20-alanine repeat allele peak and the 26-alanine repeat allele peak. The 26-alanine repeat allele peak shows a marked decrease in amplitude as compared with the 20-alanine repeat allele peak, indicating mosaicism (mutant allele determined to be 18% concentration (36% mosaic). Column II. Results of sequence analysis in 20/26 proband and mosaic carrier. A, Mutant allele sequence of the proband with full heterozygous 20/26 mutation can clearly be determined. B, Mutant allele sequence mosaic carrier is indeterminable. C, With an increased amplitude such that the tops of the peaks are no longer shown in the figure, and using the proband sequence as a reference, the mutant allele sequence of the 18% mutant allele concentration mosaic carrier can be detected. However, targeted mutation analysis would be needed for confirmation.Validation of Experimental ResultsTwo families with both a PHOX2B expansion mutation mosaic carrier parent and a heterozygous proband child were tested using both the PHOX2B-targeted mutation analysis and the PHOX2B sequence analysis tests to validate the results of the dilution sample experiments. Family 1 included a female proband, genotype 20/27, who inherited the 27-alanine repeat expansion from a mosaic carrier (mother). Family 2 included a male proband, genotype 20/26, who inherited the 26-alanine repeat expansion from a mosaic carrier (father). As expected, based on the above-described experiment using dilution samples, the PHOX2B-targeted mutation analysis was able to clearly identify the mosaic carrier in families 1 and 2 and to determine concentrations of 9% and 18% mutant allele (18% and 36% mosaic), respectively (Fig. 5). Also in line with these experimental results, the PHOX2B sequence analysis was unable to clearly show the sequence of the mutant allele in the mosaic carrier of family 1 (9% mutant allele concentration), whereas the sequence of the mutant allele of the mosaic carrier in family 2 (18% mutant allele) was determined using the proband's sequencing result as a reference sequence (Fig. 5).DISCUSSIONExpansion of the trinucleotide repeat regions is a common disease-causing mechanism and PCR amplification and analysis assays are common clinical testing methods for confirmation of many of these diseases. In the GC-rich polyglutamine and polyalanine repeat expansion diseases, preferential PCR amplification of the shorter DNA fragments has been shown to cause ADO,4,9,14,15 leading to a missed identification of the expanded allele. Mosaicism of the expanded allele has recently been identified in several of these diseases, and will further increase the risk of ADO.7,11,16–19 However, detection of mosaicism by different methodologies in trinucleotide repeat expansion disorders has not been extensively investigated. Our study examined the capacity of 2 clinical diagnostic testing methods to detect mosaicism using the
CCHS-defining PHOX2B polyalanine expansion mutations as a representative model testing system for a GC-rich trinucleotide repeat expansion disorder. Results showed that PHOX2B testing using the targeted mutation analysis had a lower LOD of mutant allele compared with PHOX2B testing using the sequencing analysis.Results of the study indicate a considerable difference in the LOD of mosaicism in PHOX2B expansion mutations between the testing methods, wherein LOD refers to the smallest amount or concentration of analyte that can be distinguished from the background at a stated confidence level (www.CLSI.org, accessed on: April 3, 2010). Although often used interchangeably, LOD and analytic sensitivity are not the same.20 According to the American College of Medical Genetics, analytic sensitivity is the proportion of biological samples that have a positive test result or known mutation and that are correctly classified as positive (www.ACMG.net, last accessed March 3, 2010). The analytic sensitivity of the PHOX2B-targeted mutation analysis, using amplification and fragment analysis, is approximately 95%,7 whereas the analytic sensitivity of the PHOX2B sequence analysis, consisting of amplification and sequencing of all PHOX2B coding regions and intron-exon boundaries, is greater than 99% (www.genetests.org). However, analytic sensitivity does not take into account the relative quantity of wild type and mutated allele within those biological samples. This relative quantity of alleles is of particular importance when considering mechanisms of testing in diseases in which mosaicism has been identified in the carriers and/or probands. Therefore, the analysis and determination of LOD in testing methods for such diseases should be determined.These results support published findings indicating that a major limitation of sequencing, in general, is the relatively high LOD and risk of ADO.21–24 In contrast, targeted mutation analysis had an extremely low LOD (1%) of mosaicism in this study. PHOX2B sequencing had a higher LOD (20%), which is consistent with the expected LOD of dideoxy sequencing assays.23 Although it is conceivable that further optimization of these assays could improve the LODs, dideoxy sequencing is limited by the fact that the lesser allele must be distinguished from the “noise” of the dominant allele and is therefore considered inherently less able of identifying low-level mosaicism.PHOX2B mutations are inherited in an autosomal-dominant manner, and in most patients they are considered to be of germline origin. However, the PHOX2B mutation is inherited as a result of low-level mosaicism in asymptomatic parents in 5% to 10% of
CCHS cases.3,7,11,12 Our study shows that the LOD for the PHOX2B-targeted mutation analysis is 1% mutant allele (2% mosaicism) whereas the LOD for the determination of the mutant allele sequence using the PHOX2B sequence analysis is 20% (40% mosaicism); the presence of known (familial) mutations can usually be detected at 10% (20% mosaicism) using sequencing. Therefore, targeted mutation analysis would be preferable for screening
CCHS-proband family members, provided the proband exhibits an expansion or deletion mutation in the polyalanine repeat region (∼95% of the cases). In addition, it has recently been reported that later onset
CCHS (LO-
CCHS;
CCHS presenting after the newborn period) might result from mosaicism as low as 10%.12 As we have shown, this concentration of mutant allele would likely not be detectable by the PHOX2B sequence analysis, suggesting that the use of targeted mutation analysis is imperative as an initial test for these LO-
CCHS cases. As cases of LO-
CCHS have also presented with PHOX2B missense mutations, which are not detectable using targeted mutation analysis, a combination of the PHOX2B sequence analysis and PHOX2B-targeted mutation analysis may be necessary to ultimately determine the absence or presence of a PHOX2B disease-causing mutation in LO-
CCHS cases. These results have broader applicability to clinical testing methods for diseases that can be caused by or inherited through mosaicism of an expansion or deletion variation, particularly in diseases defined by GC-rich trinucleotide repeat expansions in which ADO has proven a concern. Careful scrutiny is necessary in the determination of a clinical testing method for cases and families with these diseases.Care must be taken in broadly applying these results. Specifically (1) this study tested only a single-model system, and results may vary among other diseases and (2) the LOD for each of the 2 methods of PHOX2B testing was made subjectively, albeit by experts in the technique using clinical diagnostic methods and blinded to the sample concentration. This study was not meant to precisely determine the exact LOD for each genotype and each testing method but rather give an estimate of the relative LOD for these methods in the clinical testing of
CCHS. Each clinical laboratory is responsible for establishing the LOD for its own tests and for each type of mutation. Nevertheless, these results should act as a guide when considering clinical diagnostic testing methods for trinucleotide repeat expansion mutations and particularly for
CCHS.In conclusion, we have shown that targeted mutation analysis of
CCHS-causing PHOX2B polyalanine expansions has a superior limit of mutant allele detection as compared with sequence analysis of the same expansions. We, therefore, would recommend that all family members of patients with PHOX2B PARM mutations are screened using the targeted mutation analysis-based testing rather than sequencing-based testing to most accurately diagnose mosaicism. In cases of LO-
CCHS, a combination of these tests (if the initial test is negative) is recommended with the aim to diagnose mosaicism and identify mutations outside the region detectable using targeted mutation analysis. Independent of the method of testing, genetic counseling is recommended and a clear understanding of the performance characteristics of each test must be known to help parents make informed decisions about the risk in future pregnancies.REFERENCES1. Weese-Mayer DE, Rand CM, Berry-Kravis EM, et al. Congenital central hypoventilation syndrome from past to future: model for translational and transitional autonomic medicine. Pediatr Pulmonol. 2009;44:521–535[Context Link][Full Text][CrossRef][Medline Link]2. Amiel J, Laudier B, Attie-Bitach T, et al. Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome. Nat Genet. 2003;33:459–461[Context Link][CrossRef][Medline Link]3. 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Genet Anal Tech Appl. 1992;9:143–145[Context Link][CrossRef][Medline Link]allele dropout; limit of detection; mosaicism; trinucleotide repeat; CCHS00019606-201012000-0000600002548_2009_98_192_repetto_hypoventilation_|00019606-201012000-00006#xpointer(id(citation_FROM_JRF_ID_d2646e1060_citationRF_FLOATING))|11065404||ovftdb|SL0000254820099819211065404citation_FROM_JRF_ID_d2646e1060_citationRF_FLOATING[Full 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The mutant allele has 27-polyalanine repeats (genotype 20/27). The highest allele concentration (50%) corresponds to a germline, heterozygous mutation (100% mosaicism). Targeted mutation analysis (column I). Peaks represent the differently sized PHOX2B alleles with the normal 20-alanine allele on the left and the expanded allele on the right. Peak height and area are directly proportional to the relative amount of each allele. Although faint, the mutant allele peak is detectable at a 1% concentration. Inset A, Targeted mutation analysis—amplified. With amplification, the mutant allele peak can be clearly detected above the background noise at a 1% concentration. Sequencing analysis (column II). Each different color of peak indicates a different nucleotide. The lower amplitude peaks beginning at the ninth peak on each of the higher concentration examples show the sequence of the mutant allele. Decreasing concentrations correspond to lower mutant peaks. The mutant allele sequence cannot be read accurately at 10% mutant allele (20% mosaicism), although the presence of double bands at this concentration is detected. Inset B, Sequencing analysis—amplified. When amplification is increased, the double bands at 10% concentration can be seen clearly, and using the full heterozygous (50% concentration) sequence as a reference (see positions 11, 14, and 16), the mutant allele sequence can be determined. However, at the 5% concentration, the background noise produces peaks stronger than those of the mutant allele sequence (see positions 9, 11, and 14), preventing determination of the mutant allele sequence.Correlation of mutant allele concentration to the relative size (area within peak) of the mutant allele peak in the targeted mutation analysis compared with the size of the normal allele peak. Using 20/27 as a representative example, this figure indicates the relationship between peak size and mutant allele concentration. Peak ratio=[area of mutant allele peak]/[(area of wild type allele peak)=(area of mutant allele peak)]. Correlation of mutant allele concentration to peak size was fitted to a linear regression line (R2=0.9959).Targeted mutation analysis of several known congenital central hypoventilation syndrome-causing PHOX2B mutations with concentrations near the limit of detection. Results of the targeted mutation analysis at mutant allele concentrations of 5%, 2%, and 1% are shown for the 33 and 25-polyalanine repeat expansion mutations (genotypes 20/33 and 20/25) and a deletion mutation within PHOX2B (c722del38). Arrows show the location of the mutant allele peak for each of these genotypes. As shown, the 1% mutant allele is detected in all samples and can be easily discerned by zooming in (as shown previously in figure 1, inset A).Sequencing analysis of several known
CCHS-causing PHOX2B mutations with concentration near the limit of detection. The concentration of the mutant allele in each sample is 20% (40% mosaicism). As shown, the mutant sequence can be determined for all the samples.Testing of mosaic carriers in families with congenital central hypoventilation syndrome probands. Family 1 (upper half of the figure). Proband has heterozygous 27-alanine expansion mutation. Parent is a mosaic carrier of the same mutation. Column I. Results of the targeted mutation analysis in the mosaic carrier show both the 20-alanine repeat allele peak and the 27-alanine repeat allele peak. The 27-alanine repeat allele peak shows a marked decrease in amplitude as compared with the 20-alanine repeat allele peak, indicating mosaicism [mutant allele determined to be 9% concentration (18% mosaic)]. Column II. Results of sequence analysis in 20/27 proband and mosaic carrier. A, Mutant allele sequence of the proband with full heterozygous 20/27 mutation can clearly be determined. B, Mutant allele sequence of the mosaic carrier is indeterminable. C, Even at an increased amplitude such that the tops of the peaks are no longer shown in the figure, and using the proband sequence as a reference, the mutant allele sequence of the mosaic carrier is indeterminable. Family 2 (lower half of the figure). Proband has heterozygous 26-alanine expansion mutation. Parent is a mosaic carrier of the same mutation. Column I. Results of the targeted mutation analysis in mosaic carrier show both the 20-alanine repeat allele peak and the 26-alanine repeat allele peak. The 26-alanine repeat allele peak shows a marked decrease in amplitude as compared with the 20-alanine repeat allele peak, indicating mosaicism (mutant allele determined to be 18% concentration (36% mosaic). Column II. Results of sequence analysis in 20/26 proband and mosaic carrier. A, Mutant allele sequence of the proband with full heterozygous 20/26 mutation can clearly be determined. B, Mutant allele sequence mosaic carrier is indeterminable. C, With an increased amplitude such that the tops of the peaks are no longer shown in the figure, and using the proband sequence as a reference, the mutant allele sequence of the 18% mutant allele concentration mosaic carrier can be detected. However, targeted mutation analysis would be needed for confirmation.Comparison of <em xmlns:mrws="http://webservices.ovid.com/mrws/1.0">PHOX2B</em> Testing Methods in the Diagnosis of Congenital Central Hypoventilation Syndrome and Mosaic CarriersJennings Lawrence J. MD PhD; Yu, Min MD; Zhou, Lili MD; Rand, Casey M. BS; Berry-Kravis, Elizabeth M. MD, PhD; Weese-Mayer, Debra E. MDOriginal ArticlesOriginal Articles419p 224-231
