OBSERVATION Familial Periodic and Charcot-Marie-Tooth Disease in a 7-Generation Family

Fuki M. Hisama, MD

Background: A family with a complicated constella- Results: A novel missense mutation (Arg67Pro) in the tion of neurologic findings, including neuropathy, myo- myelin protein zero gene was identified in 2 patients with tonia, and , has been described in 4 stud- Charcot-Marie-Tooth disease, and a common missense ies in the medical literature since 1934. The underlying mutation (Thr704Met) was identified in the SCN4A gene cause of their disease has been the subject of consider- in 4 family members. We discuss the difficulties of geno- able speculation and has never been identified until now. type-phenotype correlation in this family.

Objective: To identify the molecular basis of this fami- Conclusions: These findings indicate that 2 indepen- ly’s neurologic disease. dent mutations segregating in this family are respon- sible for the puzzling clinical picture. Design: The coding regions of 6 genes that cause pe- ripheral neuropathy and regions of the muscle gene (SCN4A) were sequenced. Arch Neurol. 2005;62:135-138

YPERKALEMIC PERIODIC PA- METHODS ralysis (HYPP), also known as Gamstorp dis- ease or adynamia epi- FAMILY HISTORY sodica hereditaria, is an autosomal dominant disorder character- The family history was obtained from 6 individu- H als, available medical records were reviewed, and ized by attacks of muscle weakness pre- 4 individuals were personally examined. Even- cipitated by rest after exercise, cold, fast- tually, I found a 1954 study11 in the AMA Archives ing, or potassium ingestion.1 Hyperkalemic of and Psychiatry on 17 affected fam- periodic paralysis is caused by missense mu- ilymembersthatincludedelectromyographicand tations in the ␣ subunit of the muscle so- muscle biopsy results. This study cited genera- dium channel gene (SCN4A) on chromo- tionsIthroughIII,publishedin1934and1943.9,10 some arm 17q.1-3 Few other clinical features Clinical features in 7 generations are summarized are associated with HYPP. In 1887, Couzot in Figure 1. Participants gave written, informed reported short stature.4 Cardiac dysrhyth- consent under a protocol approved by the Yale 5 University (New Haven, Conn) School of Medi- mia has been reported ; 1 family had myo- cine Human Investigations Committee. DNA tonia, cardiac arrhythmia, and malignant samples were isolated from blood or buccal swabs 6 hyperthermia with general anesthesia. using standard methods. Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of peripheral nerve MOLECULAR ANALYSIS OF THE disorders characterized by distal limb weak- SCN4A AND CMT GENES ness, sensory loss, diminished or absent ten- don reflexes, and pes cavus. This disease is Seven common mutations (Thr704Met, classified by inheritance pattern (autosomal Gly1306Val, Thr1313Met, Leu1433Arg, dominant,recessive,orX-linked),bywhether Arg1448His, Met1592Val, and Val1589Met) in the axon or the myelin sheath is predomi- the SCN4A gene were screened using direct se- nantly affected, and by whether motor nerve quencing.13-16 Thr704Met was amplified using conduction velocities (NCVs) are slowed to primers 5Ј-GTCTTCAAGCTGGCCAAGTC-3Ј Ј Ј less than 40 m/s or are 40 m/s or greater.7,8 and 5 -AGACGATGAGGAAGGAGTGG-3 and We describe a family with a unique neu- the conditions described in the following para- romuscular presentation that includes graph for exon 2 of myelin protein zero (MPZ). Mutations were verified by sequencing both DNA HYPP, muscle cramping, CMT, and car- strands. Author Affiliation: Department diac arrhythmia. The clinical findings were Peripheral myelin protein 22 (PMP22) gene 9-12 of Neurology, Neurogenetics reported between 1934 and 1973. We duplication or deletion was negative, and 6 CMT Program, Yale University School performed molecular genetic studies to in- genes (PMP22, MPZ, 32 [CX32], early of Medicine, New Haven, Conn. vestigate the basis of their condition. growth response factor 2 [EGR2], neurofilament

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 I 12

II

1 2 3 4

III 123456 78910

IV (b) (a) 1234 567 8 9 10 11 12 13

V (a) (b) (a) (a) 12 3 4 5 6 7 8 9 10

VI 12 3 4567(a)8

VII

Periodic Paralysis Male CMT Female 1 CMT and Deceased Cardiac Arrhythmia Proband (a) Thr704Met Mutation (b) Arg67Pro Mutation

Figure 1. Pedigree of a family with Charcot-Marie-Tooth disease (CMT) and hyperkalemic periodic paralysis.

light [NFL], and periaxin [PRX]) were analyzed by sequencing ing nocturnal leg and diaphragm cramping while the entire coding region (Athena Diagnostics Inc, Worcester, singing opera. He had a history of paroxysmal atrial fi- Mass). A missense mutation in MPZ was detected. Exon 2 of MPZ brillation. At age 47 years, he developed gait difficulty was amplified using primers P0ex2-P21 (5Ј-CTTCCTCTGTATC- Ј Ј and distal leg numbness. Examination results showed dis- CCTTACTG-3 ) and P0ex2-M6 (5 -CTCCTTAGCCCAAGT- tal muscular , myotonia, occasional fascicula- TATCT-3Ј).17 Polymerase chain reaction (PCR) was performed in a 25-µL reaction containing 50 ng of genomic DNA, 50 ng of tions, reduced sensation below the middle of the calf, ab- each primer, 1.5mM magnesium chloride, 200µM deoxynucleo- sent reflexes throughout, steppage gait, and pes planus. tide triphosphates, and 1ϫ PCR Buffer II and 2.5 U AmpliTaq The median motor NCV was 28 m/s, and electromyog- DNA polymerase (Roche Molecular Biochemicals, Indianapo- raphy showed fibrillations and myotonia. His creatine ki- lis, Ind) using the GeneAmp PCR System 9700 (Applied Biosys- nase level was 822 U/L (reference range, 24-195 U/L). tems, Foster City, Calif). A modified touchdown protocol was DNA test results for , PMP22 dupli- used: 94°C for 5 minutes, 94°C for 45 seconds, annealing tem- cation, and CX32 were negative. Based on his sister’s pe- perature (3 cycles each at 65°C, 62°C, 59°C, 56°C, and 53°C) riodic paralysis and his myotonia, therapy for 45 seconds, and extension at 72°C for 1 minute (15 cycles was started, with relief of the muscle cramping. total), followed by 25 cycles at 94°C for 45 seconds, 50°C for 45 seconds, 72°C for 1 minute, 72°C for 10 minutes, and a 4°C hold.18 Patient IV:9, the father of patients V:8 and V:9, had The PCR products were purified (ExoSAP-IT; USB, Cleveland, episodes of periodic paralysis since infancy. His calves Ohio) and then were sequenced using an automated sequencer were enlarged in childhood, with foot deformities by age (ABI 377; Applied Biosystems). 10 years and multiple orthopedic procedures. Progres- sive leg weakness and muscle wasting began during his RESTRICTION DIGESTION teens. Examination results disclosed absent deep ten- don reflexes in the legs, footdrop, pes cavus, myotonic Restriction digests of MPZ PCR products were performed at 37°C response to muscular percussion, absent position and vi- with 10 U of HaeIII (New England Biolabs, Beverly, Mass) for bration sense, steppage gait, and the Romberg sign. The 2 hours and separated by agarose gel electrophoresis. An af- median motor NCV was 31.4 m/s, and electromyogra- fected patient’s sample served as a positive control. phy showed myotonia in 4 limbs. Patient V:10 was a 16-year-old boy with weekly epi- RESULTS sodes of periodic paralysis that lasted 10 to 30 minutes, beginning in the first year of life. Triggers included hot A 51-year-old man (patient V:8) was referred for famil- weather, rest after exertion, and certain foods. Myotonia ial neuropathy (Figure 1). At age 35 years, he began hav- was elicited on examination, and his reflexes were nor-

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 A B T A A C C T G A C G C T G G T G C T G C C A C C T G G C G C T A C C A G C C C G NLTLVLA TWR YQPE

G T A A C C T G A C G C T G G T G C T G G C T A C A C C T G G C C C T A C C A G C C C G NLTLVLAI TWP YQPE

C D NH Marker Control Control V:8 V:10 IV:12 IV:13 2

Arg67Pro

S S

COOH

Figure 2. Mutations in the ␣ subunit of the muscle sodium channel (SCN4A) gene and the myelin protein zero (MPZ) gene. A, Control (top) and patient V:10 (bottom) heterozygous for a C→T substitution (arrow) resulting in the Thr704Met mutation of SCN4A. B, Control (top) and patient IV:12 (bottom) heterozygous foraG→C substitution (arrow) resulting in the Arg67Pro mutation of MPZ. C, The Arg67Pro mutation in MPZ results in the gain of an HaeIII restriction site and a new band at 195 base pair (arrow). D, The MPZ protein with the location of the Arg67Pro mutation and the amino (NH) and carboxyl (COOH) termini indicated.

mal. His feet showed pes planus. The median motor NCV MPZ gene sequencing in patient V:8 showed a novel was 62 m/s, and electromyography showed myotonia. 201G→C variant, resulting in the replacement of argi- Patient IV:12 was a 51-year-old man with difficulty nine with proline (Arg67Pro). This missense mutation running in childhood who began to experience trip- is not described in the Inherited Peripheral Neuropa- ping, gait difficulty, and leg weakness in his 40s. A mag- thies Mutation Database (available at http://www.molgen netic resonance image of the lumbar spine showed promi- .ua.ac.be/CMTMutations, accessed Novmber 4, 2004); nent cauda equina and thickened nerve roots. He had substitutions that cause type I CMT are reported at nearby supraventricular tachycardia and type 1 diabetes melli- codons 62, 63, 65, and 68. Arg67Pro was present in 2 of tus. Examination showed distal weakness, atrophy and 2 family members with CMT and in 0 of 4 unaffected fam- sensory changes, areflexia, steppage gait, pes cavus, and ily members and was absent in 100 ethnically matched hammer toe deformity. His creatine kinase level was 867 controls (Figure 2). U/L (reference range, 24-195 U/L). The median motor NCV was 26 m/s, and electromyography showed spon- taneous fibrillations and positive waves. COMMENT Sequencing DNA from individuals V:8, V:9, VI:7, IV: 12, IV:13, and V:10 showed a C→T point mutation re- A growing number of paroxysmal disorders, including sulting in substitution of threonine with methionine epilepsy, hemiplegic , hypokalemic periodic pa- (Thr704Met) in the SCN4A gene in 4 of 4 individuals with ralysis, , , Andersen syn- typical HYPP and in 0 of 2 individuals with CMT. The drome, long QT syndrome, and malignant hyperther-

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 mia, have been found to result from mutations in a variety Funding/Support: This study was supported in part by of ligand-gated or voltage-gated ion channels.18-22 the Hellman Family Foundation (San Francisco, Calif) The evaluation of patient V:8 with CMT showed a fa- and the Swebelius Trust (New Haven, Conn). Dr Hisama ther with CMT and a sister with HYPP. Occam’s razor (a is a Paul Beeson Physician Faculty Scholar of the Ameri- medieval principle of parsimony that remains a pearl of can Federation for Aging Research (New York, NY). clinical medicine: among multiple explanations for a phe- Acknowledgment: I thank the family members and their nomenon, the simplest version should be chosen) would physicians, especially Janice R. Stevens, MD, Jonathan predict that in a parent and 2 of Goldstein, MD, and Edward Etkind, MD, for their par- his children results from variable expression of a single, ticipation and the following core facilities at Yale Uni- mutated gene. This was considered because SCN4A mu- versity for excellent technical support: the Pathology De- tations cause not only the prototypical HYPP but also nor- partment DNA Synthesis Laboratory and the W. M. Keck mokalemic periodic paralysis, myotonia without paraly- Foundation Biotechnology Resource Laboratory. sis, , and myotonia congenita.1,15 Patient V:8’s history of cardiac arrhythmia, myotonia, and response to acetazolamide therapy are consistent with a . However, he does not carry the REFERENCES Thr704Met mutation. Furthermore, peripheral neuropa- thy has not been reported in any other family with HYPP, 1. Lehmann-Horn F, Rudel R. Hereditary nondystrophic myotonias and periodic and the full pedigree showed that some members had only paralyses. 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