Genetic Investigation of Secondary and Atypical

Periodic Paralysis

Jack TarletonFullerton Genetic Laboratory

Mission Health SystemAsheville, NC

Primary = familial, or if there is no family history, a known ion channel gene mutation is found in the patient. Clinical findings similar to most PP patients.

Secondary = PP found in a patient with a disorder unrelated to ion channel impairment. No ion channel variant present. Examples: (1) PP in a patient with a renal disorder (2) PP associated with hyperthyroidism

Atypical = patients with unusual clinical presentations compared to primary PP. An ion channel gene variant may or may not be present in the patient. Examples: (1) patient with a possible diagnosis of PP but additional clinical findings previously associated with RYR-1 related disorders (2) PP associated with a supposed “benign” variant in SCN4A along with a second variant in another gene

Primary versus Secondary versus Atypical PP

Whole Exome Sequencing (WES) of Patients With Atypical Periodic Paralysis (PP)

Overview: A WES study of PP patients who are negative for known pathogenic CACNA1S, SCN4A, KCNJ2, and KCNJ18 variants. Primary and atypical PP may be difficult to clinically differentiate and standard testing using Sanger sequencing methods does not detect variants outside of the four listed genes. Next Gen Sequencing may be a better approach.

Purpose: To devise a clinical variant testing strategy (single gene versus gene panel versus whole exome). For individual patients we hope to find some genetic underpinning for their symptoms.

How patients chosen? Negative screening of the four genes above and a history of episodic weakness / paralysis.

Informed Consent Obtained

Limitations: Purely a genetic study with no functional studies. We have some clinical records on all patients but ranges from minimal to extensive. Few patients have had a full diagnostic work up to include Compound Muscle Action Potential (CMAP) testing, EMG, muscle biopsy, MRI.

Thank you to the patients who agreed to participate in the study!

Patient Overview22 Studies: 11 Duo (various combinations of two family members) or trio and 11 singleton patients. Age range was from 10 to 70 but most were 20 – 55.

All patients have a reported history of partial or full paralytic / severe weakness events. Most patients had a clinical diagnosis of HypoPP associated with hypo- or normokalemia; responsiveness to oral potassium; and common triggers, but many also have atypical and complicated symptoms such as lingering weakness between episodes, ptosis, severe myalgia, vertebral issues, cardiac complications. Many patients report lidocaine insensitivity, joint laxity, and/or “brain fog” while others don’t appear to have these features.

Patients were often responsive to different medications. Carbonic anhydrase inhibitors (acetazolamide, dichlorphenamide) not effective in some patients.

If a candidate variant was identified, it was confirmed by independent Sanger sequencing and segregation analysis was performed using DNA samples from other family members.

Mechanisms Producing PP

• Leaky sodium and calcium channels – Gating Current

• Loss of KCNJ2 function produces ATS – also produces other complications such as long QT and skeletal changes

• Seemingly benign SCN4A variants that appear to be “aggravated” by modifier variants in CLCN1. Chloride conductance important for resetting resting membrane conductance.

• Reduced mitochondrial capacity related to defect in mtATP 6 / 8

• Impaired calcium release related to RYR1 variants

• Reduced sodium-potassium pump capacity related to a gating current induced by a variant in ATP1A2 (Na+/K+ ATPase)

From: Cannon 2017 Journal General Physiology 149: 1061-64

The inexcitability of the sarcolemma is well understood when the SCN4A and CACNA1S – encoded channels are impaired. Mitochondrial/Na+K+ pumps/RYR1mechanism leading to membrane inexcitability are not well understood.

Compromised Mitochondrial Function

Aure, et al. Episodic weakness due to mitochondrial DNA MT-ATP6/8 mutationsNeurology 2013. 81:1810-8

A report that homoplasmic deleterious mutations in the mitochondrial DNA MT-ATP6/8 genes may be responsible for acute episodes of limb weakness mimicking periodic paralysis.

These authors report 100% deleterious mutations in the MT-ATP6 and MT-ATP8 genes can cause episodic weakness followed by progressive motor neuropathy. The loss of function induces a mild bioenergeticdefect with oxidative stress and permanent plasma membrane depolarization that probably underlie the paralysis episodes.

The molecular mechanisms linking the mitochondrial bioenergetic defect to ionic disturbance at the level of the cell membrane remain to be understood. However, patient had dramatic improvement of episodic weakness induced by acetazolamide treatment.

Nuclear encoded proteins that affect mitochondrial function (mitochondria depletion disorders) may potentially result in symptoms of PP.

Atypical periodic paralysis and myalgia: A novel RYR1phenotype

Matthews, et al. Neurology 2018. 90: e412–e418

Source: https://www.slideshare.net/mbbs2ndbatch/lec-2-3stud

The exact mechanism by which the RYR1 variants would cause an episodically unexcitable sarcolemma is unclear. Not all patients in the study were positive for the McManis long exercise test.

The CACNA1S-associated early-onset myopathy shares features of predominantly axial weakness and ophthalmoplegia with RYR1-related myopathies and may also feature similar reductions of the Cav1.1 protein.

A novel ATP1A2 mutation in a patient with hypokalaemic

periodic paralysis and CNS symptoms

.

Nine y.o. child with hypokalaemic periodic paralysis and CNS involvement, including seizures, but without mutations in the known periodic paralysis genes

Variant identified in ATP1A2 leading to a gating current analogous to current leaks in ion channels

Seizures explained by tissue expression of ATP1A2

Castañeda, et al.: Brain. 2018. 141: 3308–3318

Prolonged Membrane Depolarization: Potential Sites For Disruption of Excitation – Contraction Coupling

1. Neuromuscular Junction (NMJ) – post-synapse end plate potential (EPP) induced by a neuronal impulse normally exceeds the threshold for a muscle action potential. (About 50 proteins. Pre- and post-synapse). Safety factor = 3 – 5 times the required minimal signal. Defects give rise to myasthenia syndromes.

2. Impulses travel from the NMJ along the muscle surface fiber (sarcolemma) and endward along the transverse tubules through the concerted action of Nav1.4 (SCN4A) and Kir2.1 (KCNJ2), where membrane depolarization induces a conformational change of the Cav1.1 (CACNA1S) calcium channel

3. Involvement of other potassium channels and sodium-potassium pumps. The Na+/K+ ATPase pump is ATP dependent.

4. The Cav1.1 conformational change activates the ryanodine receptor (RYR1), the calcium release channel of the sarcoplasmic reticulum.

5. Calcium binds to troponin which in turn shifts tropomyosin exposing myosin binding sites. Myosin binds actin and the muscle contracts. (Myofibrillar proteins? Dystrophin-associated protein complex?)

6. If the excitation-contraction coupling (ECC) is impaired anywhere along the continuum of neural impulse to contraction, the generation of muscle force may be impaired.

Patient / Family Unit Candidate VariantsPPS 1 singleton RYR1: c.10292C>T; p. A3431V

SCN4A: c. 2717G>C; p.S906T

PPS 2 / 14 cousin - cousin duo SNTB1: c.3_8 dupGGCGGT: p.V3_A4 dupLMNA: c.1930C>T; p.R644CRYR1:pathogenic in one cousin onlyPLEC: c.12131C>T; p.T4044MMYBPC3: c.2497G>A; p.A833T (one cousin)

PPS 3 singleton PLEC: c.3130C>T; p.R1044CHSPG2: c.1082C>A; p.T361NHSPG2: c.3272G>A; p.R1091QSTAC3: c.520A>C; p.N174HCACNA1A: c.6724C>G; p.R2242GCLCN1: c.899G>A; R300Q

PPS4 mother – son duo AGRN: c.44C>G; p.P15RPLEC: c.6808C>A; p.L2270IleSNTB1: c.1307C>T; p.A436V

PPS5 proband - father duo SCN4A: c.2341G>A; p.V781IleCLCN1: c.86A>C; p.H29PSNTA1: c.1088A>C; p.E363AUTRN: c.2021C>T; p.P674L

PPS6 singleton SCN4A: c. 2717G>C; p. S906TFLNC: c. 7657C>T; p.R2553WMYH7: c.115G>A; p.V39M

PPS7 singleton CAV3: c.233C>T; p.T78MPOLG: c.2209G>C; p. G737R

Patient / Family Unit Candidate Variants

PPS8 mother –daughter duo TTN: c. 83315A>T; p.N27772 I FIS1: c. 334delC; p. R112G fs*7PRRT2: c. 647C>T; P216L

PPS9 mother-daughter duo RYR1: c.11599C>T; p.R3867CKCNE2: c.130G>A; p. E44K

PPS10 singleton SCN4A: c.952T>C; p. W318RCLCN1: c.1453A>G; p. M485V

PPS11 mother-daughter duo SYNE2: c.17561T>C; p.L5854

PPS12 singleton

PPS14 PLEC: c.12131C>T; p.T4044M RYR1: c. 4711A>G; p. I 1571VRYR1: c. 10097G>A; R3366H RYR1: c. 11798A>G; Y3933C

PPS15 cousin-cousin duo CLCN1: c.2284+5C>T

PPS16 mother-son duo SCN4A: c.1796A>G; p.H599RTYMP: c.1142T>G; p.L381R

PPS17 singleton SCN4A: c.92G>T; p.R31LSCN4A: c.241G>C; p.E81QMYLK2: c284C>A; p.A95ENEFH: c.1947_1964dup18;

p.A652_K657dup

Patient / Family Unit Candidate Variants

PPS18 cousin - cousin duo CACNA1H: c.5113G>A; p.A1705TELOVL5: c.736G>T; p. G246*TRAP1: c.1339T>A; p. Y444None cousin only FLNC: c. ?; p.R1241C

PPS19 mother – son duo SCN4A: c.4484T>C; p.I1495T

PPS20 singleton FLNC: c.2500A>G; p. T834A

PPS21 proband - mother - NEFH: c.1817C>T; p.S606Ffather POLG: c.2492A>G; p. Y831C

POMGNT2: c.1716T>G; p. F572LCLCN1: c. W118G

PPS22 singleton RYR1: c.8991C>A; p.F2997L

PPS23 singleton RYR1: c.1553T>C; p.V518A

PPS24 singleton RYR1: c.11599C>T; p.R3867C

Study Findings

• 4 patients appear to have involvement of SCN4A variants (two of these patients appear to have seemingly benign SCN4A variants in combination with some modifier – W318R and H599R). One patient with two heterozygous SCN4A variants: Arg31Leu and Glu81Gln

• 6 patients have potential involvement of RYR1 variants of uncertain significance

• No patients appear to have a Na+/K+ ATPase (ATP1A2) variant

• 4 patients do not have a candidate gene variant identified

• Several patients have variants in genes that may manifest as muscle weakness

• 2 patients have multiple heterozygous variants that may affect the efficiency of the neuromuscular junction (PLEC, HSPG). Both these patients respond well to cholinesterase inhibitors or 3,4 diaminopyridine. Two other patients have variants in genes encoding proteins present at the NMJ that segregate in family studies (AGRN, PLEC, UTRN)

• 2 patients have variants in FLNC that may be presenting a complex phenotype involving myofibrillar myopathy

• One patient has a known pathogenic CAV3 along with a POLG variant of uncertain significance

• Several patients may have possible involvement of a nuclear encoded mitochondrial depletion gene variant (POLG – polymerase gamma; TYMP – thymidine phosphorylase [mitochondrial neurogastrointestinal encephalopathy syndrome])

Speculative Mechanisms

Atypical Associations: Missense Variants Affecting the Efficiency of the Neuromuscular Junction?

• Two patients with complex presentations suggestive of myasthenia including muscle weakness with ptosis, swallowing difficulties, and shortness of breath. Both extended families are segregating missense PLEC (plectin) variants. In addition, one patient has a syntrophin-beta-1 variant while the other patient has variants in HSPG (perlecan).

• Cholinesterase inhibitors, e.g. pyridostigmine (mestinon), or pre-synaptic K+ channel blockers, e.g., 3,4 diaminopyridine (3,4 DAP), significantly help symptoms.

• A third patient in a family segregating a PLEC variant and an agrin (AGRN) variant has not been treated with pyridostigmine nor 3,4 DAP.

• Do heterozygous missense mutations lower the “safety factor” of NMJ transmission? (Safety factor is the measure of excess neurotransmitter released). Could a so-called “synergistic heterozygosity” involving multiple variants acting independently be affecting NMJ transmission?

Atypical Association: Structural Proteins Involved in Myofibrillar Myopathy Disorders?

• Two patients in the study have variants in FLN-C (encoding filamin C) both having distal episodic paralysis/weakness and cardiomyopathy. Filamen C is an actin cross linking protein involved in reorganizing the actin cytoskeleton in response to signaling events. Phenotype may fit MFM type 4, a slowly progressive, late adult onset disorder. Typical onset 40-50. Associated with slowly progressive weakness making ambulation difficult. No known connection between defective structural myofibril proteins and membrane inexcitability. CMAP not performed on these two patients.

• Other genes known to be involved in MFM: DES, MYOT, LDB3, BAG3, CRYAB

• Two unpublished families with TTN (encodes titin) variants with episodic paralysis and cardiomyopathy (personal communication).

Summary

• Atypical PP presentations are often complex with a variety of possible genetic mechanisms

• A genomic testing approach is best once primary PP is excluded

• For standardizing clinical genetic studies, a consensus of which genes to target is needed

• Recommendations and guidance for variant interpretation and functional testing are needed

• Mechanisms to explain loss of membrane excitability in complex cases are lacking

• PPA sponsored case conferences?

From Fialho, et al. Handbo Clin Neurol 2018