|AMERICAN COLLEGE OF MEDICAL GENETICS
Standards and Guidelines for Clinical Genetics Laboratories
Technical Standards and Guidelines for Huntington Disease:
|Nicholas T. Potter, PhD, University of Tennessee Medical Center
Elaine B. Spector, PhD, University of Colorado School of Medicine
Thomas W. Prior, PhD, Ohio State University
These standards and guidelines are designed primarily as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these standards and guidelines does not necessarily ensure a successful medical outcome. These standards and guidelines should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical molecular geneticist should apply his or her own professional judgment to the specific clinical circumstances presented by the individual patient or specimen. It may be prudent, however, to document in the laboratory record the rationale for any significant deviation from these standards and guidelines.
Disease-specific statements are intended to augment the general ACMG Standards and Guidelines for Clinical Genetic Laboratories. Individual laboratories are responsible for meeting the CLIA/CAP quality assurance standards with respect to appropriate sample documentation, assay validation, general proficiency and quality control measures.
Background on Huntington Disease
Gene Symbol/Chromosome Locus: HD; also IT-15 gene
OMIM Number: 143100
Brief Clinical Description
Huntington disease is a neurodegenerative disease of midlife onset that produces choreic movements and cognitive decline, often accompanied by psychiatric changes. It affects approximately 1 in 10,000 individuals. Juvenile-onset HD occurs in about 5% of affected patients, is a rapidly progressive variant and presents with rigidity, spasticity and intellectual decline before the age of 20. The symptoms result from the selective loss of neurons, most notably in the caudate nucleus and putamen, and there is currently no effective treatment. For more information see the online Gene Reviews profile at www.genereviews.org.
Mode of Inheritance
Inheritance is autosomal dominant with clinical manifestations associated with expansion of a polymorphic trinucleotide (CAG) repeat. Variable penetrance for alleles carrying 36-39 repeats has been noted, but the disease appears fully penetrant when the repeat numbers are > 40.
Gene Description/Normal Gene Product
The IT-15 gene spans more than 200 kb and contains 67 exons. The encoded protein-huntingtin-consists of 3136 amino acid residues, has a molecular weight of 350 kDa, and lacks homology to any previous known proteins. The entire genomic region has been sequenced, and the CAG expansion mutation is located in exon 1. Structural analysis of the HD gene promoter region is consistent with the gene being a housekeeping gene. Although on a cellular level mutant huntingtin is widely expressed in both neural and non-neural tissue, there is regional-specific neuronal loss in the neurons in the caudate and putamen.
The inverse relationship between the CAG repeat size (at greater or equal to 40 repeats) and age of onset is unequivocal. The relationship is particularly strong for high repeat sizes (> 50 repeats). However, the repeat length does not allow precise prediction of the age of onset for any one particular patient.
HD is caused by a toxic gain of function mechanism. The gain of function could be due to either an over-activity of the normal function, or perhaps the introduction of a novel function of the protein. The pathogenic process in relation to the expansion may involve a novel interaction with other proteins or multimerization of the protein leading to large insoluble aggregates. Intranuclear inclusions of the truncated mutant protein aggregates have been identified in brains of HD patients. These alterations are ultimately associated with, but not necessarily causative of, cell death.
Listing of Mutations
Allelic heterogeneity has not been described in HD.
Ethnic Association of HD Mutation
All major ethnic groups appear to be susceptible to expansion of the HD CAG repeat.
Special Testing Considerations
Sensitivity and Specificity
CAG repeat expansion mutations account for >99% of cases of HD. Therefore tests that effectively detect and measure the CAG-repeat region of the IT-15 gene are >99% sensitive. The absence of HD pathology has not been documented in any individual with an HD allele size of > 40 CAG repeats who died, disease-free, after living up to or past the normal life expectancy. Therefore positive results (at least one allele of 40 CAG repeats or greater) are 100% specific. Allele sizes of 26 CAG repeats and lower have never been associated with an HD phenotype in the U.S. survey or in any published study. Allele sizes of 27-35 CAG repeats are rare and have not been associated convincingly with an HD phenotype, but may be mutable as described in section 4.1.2. Allele sizes of 36-39 CAG repeats have been reported in both clinically affected and clinically unaffected individuals. Thus it is not possible to determine the specificity of test when one or both of the alleles are in the 36-39 CAG repeat range.
Diagnostic Versus Predictive Testing
This test is used for both confirmatory and predictive testing. Positive results for both confirmatory and predictive testing are considered diagnostic. It is strongly suggested that predictive testing not be offered to individuals until they are at least 18 years of age. A formal multidisciplinary predictive testing protocol is offered at many sites for individuals desiring a determination of their carrier status.
This test can be used for prenatal diagnosis in both amniotic fluid cells and chorionic villus samples (CVS). DNA samples should be obtained from both parents and run simultaneously with the prenatal sample. Maternal cell contamination studies should be performed on every prenatal sample to confirm the fetal origin of the sample being tested. In some extraordinary cases simultaneous predictive and/or confirmatory testing on a parent of the fetus and the fetus itself can be carried out.
Definition of Normal and Mutation Category (Figure 1)
Normal alleles are defined as alleles with < 26 CAG repeats . These alleles are not pathologic and segregate as a stable polymorphic repeat in > 99% of meiosis. The most common normal allele lengths contain 17 and 19 CAG repeats .
Mutable normal alleles are defined as alleles with 27-35 CAG repeats, and this repeat range is often referred to as the meiotic instability range or as "intermediate alleles." These alleles have yet to be convincingly associated with an HD phenotype, but they can be meiotically unstable in sperm, and pathologic expansion of paternally derived alleles in this size range has been described [3,4]. There have been no reports of maternally transmitted alleles in this range producing offspring with affected alleles . Approximately 1.5% to 2% of the general population carry alleles in this size range [2,6]. The likelihood that transmission of an allele in this range will expand into an HD allele is dependent upon several factors, including the sex of the transmitting individual, the size of the allele, the molecular configuration of the region surrounding the CAG repeat, and its haplotype [6,7]. This risk may be as high as 6% to10% for paternal alleles carrying a CAG repeat of 35 .
HD alleles with reduced penetrance are defined as alleles with 36-39 CAG repeats. Repeat sizes in this range are often referred to as being in the reduced penetrance range. Alleles in this size range are meiotically unstable and are associated with the HD phenotype in both clinically and neuropathologically documented cases . However, in rare cases, alleles in this range have been found in elderly asymptomatic individuals [8,9]. Although the limited number of documented cases has precluded the development of empirical penetrance risks for alleles in this range, some estimates of risk can be ascertained from an examination of individual case reports [8,9].
HD alleles with full penetrance are defined as alleles with > 40 CAG repeats. While there are several reports of elderly, clinically asymptomatic HD gene carriers with 40 and 41 CAG repeats , there are no reports of the absence of HD pathology in any individual with > 40 CAG repeats. The largest expanded HD allele detected to date carries ~250 CAG repeats in a patient with juvenile-onset disease .
Mosaicism due to both mitotic and meiotic instability has been described in brain and sperm and appears to be more pronounced in juvenile-onset cases of HD associated with larger CAG expansions . However, the degree of mosaicism is not significant enough to compromise the interpretation of CAG repeat lengths determined from DNA extracted from peripheral blood lymphocytes.
Individual U.S. laboratories offering molecular diagnostic testing for HD should be in compliance with all federal and state regulations relevant to clinical laboratory operations. This includes meeting all CLIA/CAP quality control requirements. In addition, all laboratories should be active participants in annual HD proficiency testing challenges. All methodological applications should also be in compliance with the Standards and Guideline for Clinical Genetics Laboratories developed by the ACMG . Non-U.S. laboratories should be similarly compliant with their individual countries' statutory regulations governing oversight of clinical laboratories.
Several sets of primers, PCR conditions, amplicon separation and detection techniques have been published [13-17]. Regardless of the particular PCR-based strategy selected, it is important that assay conditions and post-PCR analyses be optimized to ensure the accurate and unambiguous quantitation of repeat length (Figure 2A/B). It should be noted that CAG sizing anomalies have been observed in both comparative studies of 32P incorporation methods  and in comparative post-PCR analyses utilizing agarose, capillary, and denaturing polyacrylamide gel electrophoretic methods [18-20]. As such, accurate quantitation of patient amplicon sizes should be empirically determined by comparison to appropriate external or internal standards. These could include, but are not limited to, (M13) sequencing ladders, cloned reference standards, and appropriate normal and abnormal patient controls whose sizes have been independently verified. As it is the length of the polymorphic CAG repeat alone that is associated with the HD phenotype, patient genotyping based upon the use of a single primer pair that amplifies both the CAG and the adjacent CCG repeat  is discouraged. The CCG repeat, which lies 3ʹ of the HD CAG repeat, has been shown to be polymorphic and as a result may lead to diagnostic inaccuracies for both normal and HD allele sizing.
Appropriate controls that include a range of CAG sizes should be utilized for each analysis. It is the responsibility of the laboratory to empirically determine the detection limits for their assays. While the upper limit of detection is not known, alleles carrying ~115 CAG repeats have been amplified by PCR-based methods [21, SV Sørensen, University of Copenhagen, personal communication], whereas alleles carrying greater than ~125 CAG repeats appear to be refractory to reproducible amplification [10, D Barden, University of Washington, personal communication].
Polymorphisms surrounding or within the CAG tract have been identified and have a collective frequency of >1% in patients referred for HD testing [6,7,22-26]. These nucleotide substitutions generally can be categorized into two groups: those that modify primer-annealing sites and those that result in the loss of interruption between the CAG and CCG tracts. In the first category, nucleotide changes can result in the misinterpretation of genotyping data due to an allele-specific amplification failure associated with primer mis-annealing. As such, in certain clinical circumstances "apparent homozygosity" for two normal alleles should be interpreted with caution, and the use of alternative primer pairs or other methodologies (Southern blot or sequencing) should be used to resolve any ambiguous results [22-26]. In the second category, rare A to G substitutions in the intervening 12bp segment between the CAG and CCG tracts (CAG CAG CCG CCG) can result in increased meiotic instability of the tract as well as a miscalculation of uninterrupted CAG repeat length based upon conventional calculation formulas [7,25].
Southern blot protocols are often essential for the identification of large expansions (which may fail to amplify well) associated with juvenile-onset HD, and are useful for the confirmation of "homozygous normal" genotypes . It is recommended that all laboratories consider the use of such protocols or establish a formal referral arrangement with a facility that offers this testing for those cases that require genotypic resolution.
Since 1998 it has been the intent of the CAP/ACMG Biochemical and Molecular Genetics Resource Committee to standardize the accuracy of CAG repeat quantitation. Based on the analysis of previous CAP/ACMG proficiency testing survey results for Huntington Disease, the acceptable range for sizing CAG repeats was revised in 2012 . Acknowledging the technical limitations of size analysis, the ACMG supports the following acceptable ranges for HD clinical testing and as grading criteria for the CAP/ACMG proficiency testing survey: consensus size ± 2 repeats for alleles with less than 50 repeats; consensus size ± 3 repeats for alleles with 50-75 repeats; and consensus size +/- 4 repeats for alleles with >75 repeats.
Elements considered essential to the reporting of clinical test results are described in detail in ACMG's Standards and Guidelines for Clinical Genetics Laboratories. The following additional elements should also be included in the reporting of an HD genotype.
The methodology employed to assign the genotype. If PCR methodology was used, then a description of the primer pair(s) should be included as well as the method of amplicon separation and detection. Furthermore, each report should contain a statement regarding PCR sensitivity for the detection of large expansions. If a Southern blot was required, the restriction enzyme(s) and probe (s) should be identified. Each report must state the CAG repeat length categories and descriptors currently utilized in clinical practice, and each reportable genotype should be classified and interpreted using these categorical definitions.
Each report must include the CAG repeat numbers of both alleles with the precision of sizing fulfilling the criteria recommended by the CAP/ACMG Biochemical and Molecular Genetics Resource Committee. For large alleles determined by PCR and/or Southern blot, qualifying terms such as "approximately" or "estimated" can be used but should not be written as to create any unnecessary interpretive ambiguity. All positive results should state that genetic counseling is indicated and testing is available for other at-risk family members.
The following points on alternative diagnoses may be included.
HD phenocopies represent ~3% of patients referred for molecular confirmation of a clinical diagnosis of HD [28,29], and several other loci (HDL1 and HDL2) have recently been identified [30-32]. As such, IT-15 gene expansion in these patients will be negative. Furthermore, the considerable clinical overlap between HD and dentatorubral-pallidoluysian atrophy (DRPLA) may warrant a recommendation of DRPLA gene testing in those patients referred with a clinical diagnosis of "HD" who subsequently test negative for the expansion .
Comments on the significance of mutable normal alleles and HD alleles with reduced penetrance should not be over-interpreted in the absence of specific and well documented clinical and/or family histories. Laboratories should be familiar with the published data on these CAG repeat length descriptors and be able to articulate an interpretation that balances these data with the specific patient/pedigree information provided to the diagnostic laboratory at the time of testing [6-9, 34-35].
Diagram of IT-15 gene and the location of the polymorphic CAG repeat within exon 1. Boundaries denote CAG repeat length categories and descriptors.
PCR-based detection of the polymorphic CAG repeat utilizing 32P-dCTP incorporation and the A1/C2 primer pair (A)  or the HD1/HD3 primer pair (B) . Separation and sizing of alleles was performed on 6% denaturing polyacrylamide gels followed by autoradiography. Allele sizes are (A): 17/44; 17/17; 17/18; 17/44; 17/25; 19/45; 15/19; 15/40; 22/45; 20/24; 15/45; 16/17; 17/43; 18/45 for patients 1-14 respectively. (B): 19/43; 17/39; 19/44; 17/51; 17/39; 19/44 for patients 1-6 respectively. (Autoradiographs provided by NT Potter, University of Tennessee Medical Center)