This page copies a pamphlet produced by Athena Diagnostics. Last updated: 4/19/1995.

Athena Diagnostics

CMT Type 1A DNA Test

Athena Diagnostics, formerly Genica Pharmaceuticals Corporation, is located at Four Biotech Park, 377 Plantation Street, Worchester, MA 01605. They can be reached by calling 1-800-394-4493.

Table of Contents

Charcot-Marie-Tooth: CMT type 1A DNA test

Overview

Charcot-Marie-Tooth (CMT) disease.[1,2], also known as Hereditary Motor and Sensory Neuropathy (HMSN), is the most common inherited disorder of peripheral nerves.[3,4,5] CMT Type 1A, which accounts for the majority of all CMT, is associated with a duplication of a specific region of the short arm of chromosome 17.[6,7] The duplication has been identified in 70-90% of patients with clinical disease, making it a valuable diagnostic marker for CMT1A.[5-15] Detection of the CMT1A DNA duplication can now be readily accomplished via a blood sample analysis obviating the need for more invasive studies such as nerve conduction velocity determination and sural nerve biopsy.

Athena Diagnostics is pleased to be the first commercial laboratory to offer the CMT1A DNA Duplication Detection Test. The test utilizes a highly informative DNA detection technique, pulsed-field gel electrophoresis (PFGE), to detect the chromosome 17 DNA duplication. This test was developed at Baylor College of Medicine by Drs. James Lupski, Pragna Patel and their colleagues, and offers the highest level of accuracy currently available.[6,8]

Detection of the CMT1A duplication has important implications for diagnosing patients with inherited as well as sporadic peripheral neuropathies. In addition to its identification in patients with familial CMT1,[6-14] spontaneous CMT1A mutations have been found in many sporadic CMT patients.[7,8,15] The CMT1A DNA Test is thus a highly specific diagnostic marker of disease which can supplement or replace the electrophysiologic tests currently in use. The CMT1A DNA Test has further clinical applications in genetic counselling of patients and at-risk family members, as well as in prenatal diagnosis.

Any patient who has a chronic idiopathic peripheral neuropathy, regardless of family history, is a candidate for the CMT1A DNA Test.

Figure 1 The 17p karyogram. The bold lines represent the CMT1A duplication.[3]

Clinical reasons for performing the test

Charcot-Marie-Tooth (CMT) polyneuropathy syndrome, or the hereditary motor and sensory neuropathies (HMSN I, II, and III) are a genetically and clinically heterogeneous group of disorders of the peripheral nerves characterized by an insidious onset and slowly progressive weakness of the distal muscle with mild sensory impairment.[3,4] The clinical manifestations can vary considerably in severity and age of onset, although patients typically present during late childhood or early adulthood. Clinical findings associated with CMT1 include:
  1. Toe walking in early childhood.
  2. Slowly progressive distal muscle atrophy and weakness.
  3. Deformities of feet and hands (pes cavus, claw hand).
  4. Gait disturbance due to dorsiflexor weakness(slapping gait).
  5. Absent or decreased deep tendon reflexes.
  6. Nerve hypertrophy with onion bulb formation on sural nerve biopsy.
  7. Symmetrically-decreased nerve conduction velocity (NCV<40m/sec).
The molecular test for CMT1 A is designed to assist in the diagnosis of any patient who has a chronic idiopathic peripheral neuropathy, regardless of whether there is a family history. Any child or adult who has features of a predominantly motor neuropathy, with or without foot deformity, and for whom there is a clinical suspicion of Chronic Idiopathic Demyelinating Polyneuropathy (CIDP) should be tested. All patients at risk for CMT, that is, those who have one affected parent or sibling, should also be tested. Asymptomatic, at-risk patients with normal neurologic examination may have the duplication along with slow nerve conduction studies. The test can thus be instrumental in genetic counselling and in presymptomatic and prenatal diagnosis.

The CMT1A duplication is diagnostic of Charcot-Marie-Tooth type 1A.[6-17]. No false positive occurrences have been identified. to date. Although electrophysiologic tests are diagnostic of a hereditary neuropathy, these tests do not have the specificity of the molecular test.

All patients with either a positive family history or a symmetrical polyneuropathy of unknown origin are candidates for the CMT1A DNA Test.

The CMT1A duplication

CMT1A is associated with a submicroscopic 1.5 million base pair tandem DNA duplication on chromosome 17.[17,18] The CMT1A duplication has been identified in multiple CMT1A families of varied ethnic origins, in multiple unrelated CMT1 patients and in sporadic (de novo) patients.[6-17] It is therefore a valuable biological marker for CMT1A in many pedigrees and is useful in the clinical evaluation of patients with idiopathic polyneuropathies such as chronic idiopathic demyelinating polyneuropathy (CIDP). In several studies, 70-90% of clinically-diagnosed CMT1A patients had the duplication.[6-17]

The PMP22 gene, which encodes a peripheral nerve myelin protein, has been mapped within the CMT duplication, and has been proposed as the CMT1A candidate gene.[19-22] This was recently confirmed by the identification of PMP22 point mutations in rare nonduplication patients with clinical CMT1A.[23,24] The majority of CMT patients, however, have the CMT1A duplication.

A DNA deletion associated with Hereditary Neuropathy with Liability to Pressure Palsies(HNPP) has recently been mapped to chromosome 17p11.2, and includes all markers that are known to map within the CMT1A duplication.[25]

Figure 2 shows a representative autoradiograph from a patient with the CMT1A duplication specific junction fragment compared with a normal individual.

Test Methodology

Athena Diagnostic's test for CMT1A is a molecular test which identifies the chromosome 17 duplication using the most informative DNA detection method currently available.

The test was developed at Baylor College of Medicine by Dr. James Lupski and colleagues, who first reported the association of CMT1 with a chromosome 17 duplication.[6]

Patients' white blood cells are separated and carefully embedded in agarose plugs prior to processing the DNA, thus minimizing the shear stress of DNA extraction techniques. Restriction endonucleases are diffused into the agarose-embedded patient DNA and pulsed-held gel electrophoresis then is utilized to separate large segments of DNA containing CMT1A duplication-specific junction fragments. These junction fragments are then detected by hybridization with a CMT1A duplication specific probe (CMT1A-REP).[18] This probe identifies the homologous regions that flank the CMT1A duplication monomer unit.[18]

Test results and interpretation

The presence of the 500kb CMT1A duplication specific junction fragment indicates a positive result and is diagnostic for Charcot-Marie-Tooth type 1A (figure 2). These patients should be referred for genetic counselling.

A negative result is indicated by the absence of the 500kb CMT1A duplication specific junction fragment (figure 2).

The CMT1A duplication has been identified in 70-90% of patients with clinical CMT1.[6-17] Rare point mutations in the peripheral nerve myelin protein gene, PMP22, have recently been identified in some CMT1A patients who did not exhibit the duplication.[23,24] A negative result therefore does not rule out a diagnosis of Charcot-Marie-Tooth disease.

Case reports - Case 1

History

This 10 year old boy complained of leg cramps and clumsiness. His motor development was normal. He was in the fifth grade and was performing academically well. At age 7 he began to walk on his toes. He sprained his right ankle twice in the last year. He also complained of hand cramps after long periods of writing. His maternal grandfather had "weak feet". His maternal uncle wears leg braces. His mother had no complaints except for occasional leg cramps after long walks. Two younger siblings had no neurologic complaints.

Examination

He appeared healthy. His gait showed that he walked on his toes and was unable to walk on his heels. His heel cords were tight. There was weakness (4/5) of the dorsiflexors of both feet. The legs showed mild atrophy of the anterior tibialis and peroneal muscles. There was no atrophy or weakness of the intrinsic hand muscle. The stretch reflexes were absent in the upper and lower limbs. The plantar responses were flexor. There was decreased pricking sensation distally in stocking distribution. The great auricular nerves were enlarged on both sides but the left one was more visible. When palpated, the ulnar and peroneal nerves were found to be enlarged. He had mild pes cavus deformity.

Differential diagnosis

The patient has a chronic. predominantly motor hypertrophic polyneuropathy. The most likely diagnosis is chronic inflammatory demyelinating polyneuropathy (CIDP). The hereditary nature of the disease was confirmed by examining his mother, who was mildly obese, could not walk on her heals, and was areflexic. Her NCV's were 24.7 m/s in both median and ulnar nerves without multifocal blocks. The family history suggests an autosomal dominant inheritance pattern.

Key laboratory tests

Molecular tests showed that the CMT1A duplication was present in the patient, his mother, and a younger brother. Motor nerve conduction velocities were performed. The motor NCV of the right peroneal nerve was 14.7 m/s; the motor NCV of the left ulnar nerve was 16.9 m/s; and the motor NCV of the right median nerve was 21.2 m/s. He had motor distal latencies that were proportionately prolonged and absent sensory nerve action potentials. There was no electrophysiologic evidence of motor conduction blocks or abnormal temporal dispersion. Lack of conduction blocks argues against CIDP.

Diagnosis

Charcot-Marie-Tooth type 1A Polyneuropathy.

Treatment and course

Physical therapy to stretch the heel cords and ankle foot orthosis (AFO) to improve his gait.

Case reports - Case 2

History

This 34 year old male has had high arches and hammer toes since his teenage years. His ankles would turn easily as a child, however, he could rollerskate, but not well. In his late twenties he noted the sensation of coldness in his feet, which was greater in the left than in the right. It has become worse since then. Lately he has noticed decreased grip strength in his hands; he has difficulty opening jars, buttoning and unbuttoning his shirts, and using zippers. His ankles have gotten thinner and he has noticed a change in his gait. His paternal grandfather had high arches and hobbled on his heels when he walked. His father had high arches and thin ankles and one of this brothers had high arches. One daughter is unable to walk on her heels. He denies any history of diabetes mellitus, denies drinking, and has not been exposed to toxic materials. There is no history of autonomic dysfunction.

Examination

His general appearance is normal. He was unable to walk on his heels and had a slapping steppage gait. He had pes cavus and atrophy and weakness of leg muscles, primarily the anterior tibialis, toe extensors and peroneal muscles. Both hands show atrophy of the thenar and hypothenar muscles, wasting of interossei and difficulty with finger extension. He had decreased reflexes throughout, but absent ankle and patellar responses. There was decreased sensation distally in the legs in a stocking distribution. He had palpable enlarged nerves.

Differential diagnosis

The patient has a chronic predominantly motor hereditary polyneuropathy. Chronic inflammatory demyelinating polyneuropathy (CIDP) is unlikely because of the NCV, which shows no evidence of multifocal blocks. Absence of heavy metals rules out most of the important toxic causes. B12, folate and complete blood count were normal. Anti-GM1 antibodies were normal.

Key laboratory tests

Molecular tests showed that the CMT1A duplication was present in the patient, his brother and his daughter. The right median motor NCV was 29 m/s. Responses were unelicitable from the peroneal nerve, and the motor NCV of the right ulnar nerve was 24 m/s. He had proportionally long distal latencies and absent or low sensory nerve action potentials.

Diagnosis

Charcot-Marie-Tooth type 1A Polyneuropathy

Treatment and course

Ankle foot orthosis (AFO) improved his gait and decreased his leg cramps. Occupational therapy provided him with tools to button his shirts, pliers for zippers, and tools to open jars and door knobs.

Handling and shipping requirements

Two filled 8.5ml yellow top tubes containing Acid Citrate Dextrose (ACD) solution A shipped on the same day blood is drawn are required. Ship overnight at room temperature. The samples must arrive at Athena Diagnostics within 48 hours after blood is drawn.

DO NOT REFRIGERATE OR FREEZE SAMPLES

Canadian and overseas accounts require special courier assistance. Please call Technical Services at 508-756-2886 or FAX at 508-753-5601.

CMTA

The Charcot-Marie Tooth Association is a non-profit (501-C-3) organization devoted to the CMT patient family community and the medical/scientific community who treat them. The CMTA has a program of education and support through its quarterly newsletter, conferences, videotapes, regional support groups and other informative literature. For further information, you may contact the CMTA at 600 Upland Ave, Upland, PA 19015 USA (610-499-7486).

References

  1. Charcot J-M, Marie P: Sur une forme particulaiere d'atrophie musculaire progressive souvent familiale debutant par les pied et les jambes et atteignant plus tard les mains Rev Med; 6:97-138.
  2. Tooth HH: The Peroneal Type of Progressive Muscular Atrophy. London: 1886.
  3. Lupski, JR, Garcia CA, Parry G, Patel: Charcot-Marie-Tooth polyneuropathy syndrome: Clinical electrophysiological and genetic aspects. In Appel S (ed.) Current Neurology . Vol 11. Chicago, Mosby Yearbook 1991; pp.1-25.
  4. Dyck PJ, Chance PF, Lebo RV, Carney JA: Hereditary motor and sensory neuropathies. In Peripheral Neuropathy, Third Edition, PJ Dyck, PK Thomas. JW Griffin, PA Low and JF Podulso, (eds.) Philadelphia: W.B. Saunders 1992: pp.1094-1136.
  5. Skre H: Genetic and clinical aspects of Charcot-Marie-Tooth disease. Clin Genet 1974 3; 6:98-118.
  6. Lupski JR, Montes de Oca-Luna R, Slaugenhaupt S, et al: DNA duplication associated with Charcot-Marie-Tooth disease type 1A. Cell 1991; 66:219-232.
  7. Raeymaekers P, Timmerman V, Nelis E, et al: Duplication in chromosome 17p11.2 in Charcot-Marie-Tooth neuropathy type 1A (CMT1 A). Neuromusc Dis 1991; 1 :93-97.
  8. Wise C A, Garcia CA, Davis S, et al: Molecular analyses of unrelated Charcot-Marie-Tooth disease patients suggest a high frequency of the CMT1A duplication. Amer J Hum Genetics , in press.
  9. Raeymaekers P, Timmerman V, Nelis E, et al: Estimation of the size of the chromosome 17p11.2 duplication in Charcot-Marie-Tooth neuropathy type 1 A (CMT1A). J Med Genet 1992; 29: 5-11.
  10. Hallam PJ, Harding AE, Berciano J, Barker DR, Malcom S: Duplication of part of chromosome 17 is commonly associated with hereditary motor and sensory neuropathy type 1 (Charcot-Marie-Tooth disease type 1). Ann Neurol 1992: 31:570-572.
  11. MacMillan JC, Upadhyaya M, Harper PS: Charcot-Marie-Tooth disease type 1 A. (CMT1A): Evidence for trisomy of the region p11.2 of chromosome 17 in South Wales families. J Med Genet 1992 p.29:12-13.
  12. Chance PF, Matsunami N, Lensch MW, Smith BS, Bird TD: Analysis of the DNA duplication 17p11.2 in Charcot-Marie-Tooth neuropathy type 1 (HMSN1) pedigrees: additional evidence for a third autosomal CMT1 locus. Neurology 1992; 42:2037-2041.
  13. Bellone E, Mandich P, Mancardi GL, Schenone A, Uccelli A, Abbruzzese M, Sghirlanzoni A, Pareyson D, Ajmar F: Charcot-Marie-Tooth (CMT) lA duplication at 17p11.2 in Italian families. J Med Genet 1992; 29:492-493.
  14. Brice A, Ravise N, Stevanin G, et al: Duplication within chromosome 17p11.2 in 12 families of French ancestry with Charcot-Marie-Tooth disease type 1 A. J Med Genet 1992; 29:807-812.
  15. Hoogendijk JE, Hensel GW, Gabreels-Festen AAWM, et al: De Novo mutation in hereditary motor and sensory neuropathy type 1. Lancet 1992; 339:1081-1082.
  16. Lupski JR: An inherited DNA rearrangement and gene dosage effect are responsible for the most common autosomal dominant peripheral neuropathy: Charcot-Marie-Tooth disease type 1 A. Clin Res 1992; 40:645-652.
  17. Lupski JR, Liu P, Williams LL, Patel PI: Stable inheritance of the CMT1A DNA duplication in two patients with CMT1 and NF1. Am J Med Genet l 993; 45:92-96.
  18. Pentao L, Wise CA, Chinault AC, Patel Pl, Lupski, JR: Charcot-Marie-Tooth type 1A duplication appears to arise from recombination at repeat sequences flanking the 1.5 Mb monomer unit. Nature Genetics 1992; 2:292-300.
  19. Patel Pl, Roa BB, Welcher AA, et al: The gene for the peripheral myelin protein PMP-22 is a candidate for Charcot-Marie-Tooth disease type 1 A. Nature Genetics 1992; 1: 159-165.
  20. Valentijn LJ, Bolhuis PA, Zorn I, et al: The peripheral myelin gene PMP-22/GAS-3 is duplicated in Charcot-Marie-Tooth disease type 1A. Nature Generics 1992; 1:166-170.
  21. Timmerman V, Nelis E, Van Hul W, et al: The peripheral myelin protein gene PMP-22 is contained within the Charcot-Marie-Tooth disease type 1A duplication. Nature Generics 1992; 1:171 -175.
  22. Matsunami N, Smith B, Ballard L, et al: Peripheral myelin protein-22 gene maps in the duplication in chromosome 17p11.2 associated with Charcot-Marie-Tooth 1A. Nature Generics 1992; 1:176-179.
  23. Valentijn LJ, Baas F, Wolterman RA, et al: Mutations of PMP-22 in trembler J mouse and Charcot-Marie-Tooth disease type 1A. Nature Genetics 1992; Vol 2:288-291.
  24. Roa BB, Garcia CA, Suter U, et al: Charcot-Marie-Tooth Disease type 1A associated with de novo point mutation in the PMP22 gene. N. Engl. J. Med. 1992; 329, 96-101.
  25. Chance PF, Alderson MK, Leppig KA, et al: DNA deletion associated with hereditary neuropathy with liability to pressure palsies. Cell 72, 143-151, 1993.