In this chapter, we describe in detail our implementation of FISH technologies in the diagnosis of three myelinopathies: two neuropathies of the peripheral nervous system (PNS), Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) and one leukodystrophy of the central nervous system (CNS), Pelizaeus-Merzbacher disease (PMD), in which either duplication or deletion of a large DNA sequence with concomitant gene-dosage alteration has been implicated.
These disorders present with specific diagnostic challenges, in particular, the genomic duplications. The duplications found in CMT1A and PMD are not resolvable on the metaphase chromosome. Therefore, the duplication analysis must be performed on interphase nuclei to allow for the separation of FISH signals so that the duplications can be visualized. Additionally, the laboratory performing the analysis must recognize the statistical distinction between replication of a locus (DNA synthesis during the cell cycle) and true, pathologic genomic duplication. However, these challenges can be met and a reliable FISH-based assay can be developed and implemented in the diagnostic laboratory.
Inherited neuropathies are a heterogeneous spectrum of disorders with sometimes overlapping clinical features (10). Essentially, every mode of Mendelian inheritance pattern (autosomal dominant, recessive, and X-linked), as well as sporadic cases, have been described for inherited neuropathies (10,11). However, even within a particular mode of inheritance there is considerable variation in clinical severity among, not only unrelated individuals, but also affected family members including affected identical twins (10). The heterogeneous nature of these neuropathies and the fact that they sometimes share clinical features with acquired conditions have often posed a diagnostic dilemma to the clinician. However, advances made in the last decade have significantly improved the ability to correctly diagnose these neuropathies. Consequently, patients who present with these conditions can now receive more appropriate genetic counseling and a more precise prognosis.
220.127.116.11. Clinical Presentation and Molecular Genetic Analysis
Charcot and Marie (12) and, independently, Tooth (13), described an unusual form of progressive muscular atrophy characterized by slow progression of symptoms with initial involvement of the feet and legs, often followed years later with involvement of the hands. Both studies highlighted the inherited nature of the disease well before Men-delian laws of inheritance were rediscovered and the so-called Charcot-Marie-Tooth disease eventually became recognized as the most common inherited disorder of the PNS, affecting one person in 2500 (14). With this rate of incidence, CMT is also one of the most common genetic disorders. Tooth correctly classified the disorder as a neuropathy, and not a myelopathy, as was postulated by Charcot and Marie. Characteristically, the illness presents within the first two decades of life with early symptoms including complaints of frequent tripping and ankle-sprains and an equine-like gait resulting from footdrop. Progressive muscle atrophy of the distal muscles can lead to a stork-leg appearance in some patients. Pes cavus deformity, although usually not seen early in the course of the disease, eventually develops and seems to progress with age. Weakness of the intrinsic hand muscles usually occurs late in the course of the disease with severe cases resulting in claw-like deformities (15).
Two major forms of CMT are delineated based on disease pathology and electro-physiology (15,16). Type 1 CMT (CMT1) is characterized by a severely slowed motor nerve conduction velocity (NCV), usually less than 40 m/s (17), and onion bulb structures evident upon sectioning of the peripheral nerve (18). The clinical features of CMT1 appear to arise from myelinated fiber loss and denervation and it is referred to as the hypertrophic form (18). In contrast, type 2 CMT (CMT2), retains an essentially normal NCV (otherwise only mildly reduced) with decreased amplitudes and onion bulb structures are seen only occasionally (18). The clinical features of CMT2, for the most part, result from axonal dysfunction. Hence, CMT2 is referred to as the neuronal form. CMT1 can be further subcategorized, based on genetic linkage and the gene involved, into autosomal dominant CMT1A (PMP22 in 17p12) (19) and CMT1B (MPZ in 1q21.2-q23) (20), and X-linked dominant CMTX (Cx32 in Xq13.1) (21). Although likely to be less frequent, an autosomal recessive form of CMT has also been documented (15). However, while multiple modes of inheritance have been documented for CMT, it is the autosomal dominant CMT1A that is the most common form of the disease. Furthermore, it is CMT1A and its unique molecular genetic relationship to another clinically distinct demyelinating neuropathy, HNPP, which has revealed a novel disease-causing mechanism in humans and which has led to the first Mendelian disorder to be diagnosed by FISH (22).
Originally described in a family with recurrent peroneal neuropathy over multiple generations (23), the clinical presentation of HNPP is usually much less severe than CMT with periodic episodes of numbness and muscular weakness that follow relatively minor compressions or trauma to the peripheral nerves (24). Patients with HNPP sometimes show mildly slowed NCV. Peripheral nerve biopsies show segmental demyelina-
Fig. 2. Schematic representation of CMT1A duplication, HNPP deletion; and PMD duplication with accompanying FISH images from representative patient test samples. (A) Ideogram of G-banded 17p homologs from a typical CMT1A patient. In the normal homolog, the 1.5 Mb chromosomal region flanked by the CMT1A-REP (black box) is shown and includes a single copy of the PMP22 locus (gray box) as detected by a digoxigenin/rhodamine labeled probe (red circle). A single copy of the FLI control locus is detected by a biotin/FITC labeled probe (green circle). The homolog with the cryptic duplication at the PMP22 locus contains three copies of the CMT1A-REP, two copies of the PMP22 gene and spans 3.0 Mb. A single copy of the FLI control locus is detected. (B) Typical results from of a FISH test on an interphase nucleus from peripheral blood of a CMT1A patient. The red signal represents hybridization of the test probe to the PMP22 locus and the green signal hybridization of the control probe to the FLI locus. (C) Ideogram of G-banded 17p homologs from a typical HNPP patient. The normal homolog is essentially the same as that of the CMT1A patient, but
Fig. 2. Schematic representation of CMT1A duplication, HNPP deletion; and PMD duplication with accompanying FISH images from representative patient test samples. (A) Ideogram of G-banded 17p homologs from a typical CMT1A patient. In the normal homolog, the 1.5 Mb chromosomal region flanked by the CMT1A-REP (black box) is shown and includes a single copy of the PMP22 locus (gray box) as detected by a digoxigenin/rhodamine labeled probe (red circle). A single copy of the FLI control locus is detected by a biotin/FITC labeled probe (green circle). The homolog with the cryptic duplication at the PMP22 locus contains three copies of the CMT1A-REP, two copies of the PMP22 gene and spans 3.0 Mb. A single copy of the FLI control locus is detected. (B) Typical results from of a FISH test on an interphase nucleus from peripheral blood of a CMT1A patient. The red signal represents hybridization of the test probe to the PMP22 locus and the green signal hybridization of the control probe to the FLI locus. (C) Ideogram of G-banded 17p homologs from a typical HNPP patient. The normal homolog is essentially the same as that of the CMT1A patient, but tion and remyelination with tomaculous or sausage-like focal thickenings of the myelin sheath (25). While carpal tunnel syndrome and other entrapment neuropathies are frequent manifestations of HNPP, in severe cases, the clinical presentation can resemble that of CMT1A.
Molecular genetic analysis has demonstrated that DNA rearrangements have been implicated in the majority of patients with either CMT1A or HNPP: a 1.4Mb tandem DNA duplication in 17p12 (22,26,27) associated with CMT1A and the reciprocal 1.4Mb deletion in 17p12 (28) associated with HNPP. The 1.4Mb genomic region, which is either duplicated in CMT1A or deleted in HNPP, is flanked by a large 24kb direct repeat termed CMT1A-REP (26,29). The identification of the CMT1A-REP in conjunction with genotype analysis of de novo patients (30), was immediately suggestive of an unequal crossing-over mechanism that might explain the duplication and deletion observed in CMT1A and HNPP, respectively. Subsequently, molecular studies firmly established that the CMT1A duplication and the HNPP deletion were indeed products of a reciprocal recombination (31) involving a recombination hot spot (32,33) within the CMT1A-REP. Combined with the mapping of the peripheral myelin protein-22 gene (PMP22) to the duplicated region in17p12 (34-36), a molecular understanding of the etiology and pathology of CMT1A and HNPP was revealed. The PMP22 gene became the first and best example of a dosage-sensitive gene, which in the majority of CMT1A patients is present in three copies, while in the majority of HNPP patients, is present in one copy. The imbalance of the PMP22 gene copy number ultimately results in a disturbance of the development or maintenance of myelin with the concomitant phenotype of patients with either of these neuropathies (11,37).
1.2.2. CMT1A and HNPP
18.104.22.168. FISH-Based Strategy for the Diagnosis of CMT1A and HNPP
The chromosomal aberrations associated with CMT1A (the gain of a 1.4Mb genomic region in 17p12) and HNPP (the reciprocal loss of a 1.4 Mb genomic region in 17p12) (see Fig. 2) are both cryptic aberrations and are undetectable on routine G-banded chromosomes. However, knowledge of the gene implicated in the disease phenotype (e.g., PMP22), identification of a locus close to, but completely excluded from the micro
Fig. 2. (continued) in the homolog with the cryptic deletion, The PMP22 locus has been deleted and only one copy of the CMT1A-REP remains. A single copy of the FLI locus is retained. (D) Typical results from both an interphase nucleus and metaphase chromosomes (insert) from a patient with HNPP. The white arrows in (B) and (D) point to the duplicated and deleted PMP22 locus in the CMT1A and HNPP patients, respectively. (E) Ideogram of G-banded Xq homologs from a typical PMD patient with duplication of the PLP locus. A single copy of the PLP locus is detected by a digoxigenin/rhodamine labeled probe (red circle) and a single copy of the BTK/GLA locus (which is located about 2 Mb proximal to the PLP locus) is detected by a biotin/FITC probe (green circle). The homologue with the cryptic duplication contains two copies of the PLP locus within a genomic region which is variable in size and in most patients, one copy of the BTK/GLA locus. (F) Typical results from a FISH test on an interphase nucleus from peripheral blood of a PMD male patient. (G) FISH results on an interphase nucleus from a female carrier of a PMD duplication. The white arrows in (F) and (G) indicate the duplicated PLP loci. (See color plate 7 appearing in the insert following p. 82)
duplication/deletion (e.g., FLI in 17p11.2), and identification of cosmid contigs or PACs that are specifically mapped to either the PMP22 locus or the FLI locus, are all the requisites needed (see Fig. 1A) for the design of an effective FISH strategy to detect the duplication and deletion observed in CMT1A and HNPP, respectively (38) (see Fig. 2). Therefore, an accurate and reliable interphase-based FISH strategy can be designed for the diagnosis of both CMT1A and HNPP and has been routinely implemented in a clinical cytogenetics laboratory (Kleberg Cytogenetics Laboratory, Baylor College of Medicine, Houston, TX, USA). As mentioned earlier, since the genomic region involved in the CMT1A duplication is not resolvable on routine metaphase chromosomes, hybridization analyses need to be performed on interphase nuclei to facilitate the visualization of the duplication (22,38) (see Fig. 2B). In the case of HNPP, since it is a loss of signal that is the diagnostic criteria, analyses can be performed on either interphase nuclei or metaphase chromosomes (see Fig. 2C and D). The interphase/ metaphase-based FISH method developed for the diagnosis of CMT1A and HNPP (38) is presented in detail in Subheading 3.
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