PROTOCOLS FOR NEUROMUSCULAR GENETIC DIAGNOSIS

…the easy problems have been solved already! (mostly)

OBJECTIVES

Review the pluses and minuses of the current diagnostic tools

Illuminate some pitfalls we might be able to fall into less often

Shorten and reduce costs of the diagnostic odyssey, while enhancing our likelihood of success and benefit for our patients

Make the case for a true interdisciplinary effort in neuromuscular diagnosis

WHAT’S WRONG WITH THIS PICTURE?

Credit: Jain Foundation, LGMD2I foundation

WHAT DIFFERENCE DOES IT MAKE?

Identify the medical risks Cardiac Pulmonary Anesthesia/malignant hyperthermia Diabetes, scoliosis, HCM Retinopathy, hearing loss

Genetic counseling accuracy XLR vs AR XLSD vs AD vs mito In some cases, clinical treatments or clinical trials may be

available depending on the specific genetic diagnosis Prognosis, what to expect/what helps, support groups

THE TOOLS FOR DIAGNOSIS - $

Family history Misdiagnosis, misreport, actually getting records

Physical exam Limited by cooperation UMN vs LMN, on a good day Cerebellar versus proprioceptive ataxia Muscle bulk, contracture, facial function, EOM, winging, microcephaly

Metabolic (non-genetic lab) tests Sometimes specific and quick. Sometimes not. CPK, aldolase, TFT AA, OA, isoelectric transferrin, CoQ10, vitamins Lactate/pyruvate – lots of false + and – Alpha-fetoprotein CSF studies

THE TOOLS FOR DIAGNOSIS – $$ Electrodiagnosis

Myopathic vs neuropathic, axonal vs demyelination Kids hate it. Especially repetitive stim, which is not always clear Myotonia is not as specific a finding as you’d like and may not be there in all

“myotonic” disorders May be a quick answer for floppy baby (SMA yes or no) UMN lesion can be confounding Very specific for compressive and radicular pathology

Biopsy More likely specific especially on immunostaining or EM Pitfalls with related protein staining affected May be nonspecific myopathic or even normal in some conditions Issues with specimens for send out – research vs clinical

Muscle Imaging Fatty infiltration vs inflammation Affected vs unaffected muscles

THE TOOLS FOR DIAGNOSIS – $$-$$$ Specific single gene tests

When common things occur commonly or single best explanation fits pretty well PANELS

The good, the bad and the ugly AVOID CMTD, HSP, SCA, DM – not supposed to be “orderable” here Some cover better than WES for specific conditions

Microarrays, snp arrays Yield about 15%, towards 20% Now requires consent Regions of homozygosity, duplications, deletions

Whole Exome Requires genetics consultation Expensive and not always covered for parent Best chance for diagnosis of rare condition if Brad Schaefer does not know

what it is off the top of his head Tool for discovery of new genes

From Prevention Genetics

http://preventiongenetics.com/files/6813/8619/3468/PricelistByDisease_11-15-13.pdf

CHARCOT MARIE TOOTH DISEASE(S)

Supect: Distal weakness, cavus, hypo or areflexia, Commonly +FH, “clumsy”

Options: Edx or PMP22 dup gene test alone first Typical, PN only, no need for other screenings

If atypical at all, CMT1x, MPZ, MFN also reasonable to do Edx for slow, very slow, or normal (demyelinating, intermediate, or

axonal) Partial panel vs whole exome

DO NOT GET THE FULL PANEL 1a-f = AD, demyelinating 2a-p = AD, axonal 4a-j = AR, demyelinating, internediate or axonal X1-5 (not 3a-e) “ ”

Mito and nuclear mito (e.g. PDH), HSANs, SCAs, leukodystrophies also cause hereditary neuropathy

NERVE BIOPSY??

Dejerine-Sottas not genetically specific PMP22, EGR2 (CMTD 1D, 4E), MPZ, PRX

Vasculitic, infectious or other acquired Sural (sensory) or epidermal for small-fiber Gene test might be better for amyloid (TTN)

Portugese variant 50% Fabry, Tangier, giant axonal neuropathy Reference:

http://archneur.jamanetwork.com/article.aspx?articleid=782848

DYSTROPHINOPATHY

Suspects Global delay in a boy as well as abnormal gait Elevated “LFT” no jaundice, alk phos, GGT OK Floppy baby with contiguous gene syndrome Rhabdmyolysis

CPK 4-5 figures, not clinically a myositis Utah dystrophinopathy (or other) gene test Leiden reading frame checker Clinicaltrials.gov Biopsies?

Research purposes Negative gene test

Edx not helpful

MYOTONIA

Suspects for myotonic dystrophy Clubfoot, floppy, facial weakness, global delay May be premie and look like or have CP also Adult with cataract, DM, low energy, temporal wasting

Myotonia not always present on Edx early At ACH – get only DM1 for number of repeats NOT PANEL

Myotonia Congenita is different disease Becker/Thomsen Strength low for degree of muscle bulk, warm-up CLCN1, SCN4a

Also consider Thyroid disorders Other channelopathies (e.g. KCNJ2/Tawil-Anderson short QT) Brody myopathy (ATP2a1) Some LGMD (especially 1c)

justsayno.

ANTERIOR HORN CELL DISEASE

Edx faster, should be confirmed

Rough correlation with SMN2 copies and severity

Biopsy may also be diagnostic, with group atrophy, not often required

Other rare conditions HBSMA SMARD DSMA - UE AMC

SCA, HSP

Friedreich’s ataxia (frataxin, not fragile X) Covered by MDA; others are generally not Cardiomyopathy, diabetes, scoliosis

Unless family history positive for specific gene,

Just say WES. (Just my $0.02)

TRANSITIONS Adhalin = alpha sarcoglycan SCARMD = sarcoglycanopathy or other LGMD2

High CPK with some dystrophin on biopsy ≠ Becker’s In 2006 had six genes for dystroglycanopathy, 50%

undiagnosed, now about 15 and fewer Some conditions caused by more than one gene Some genes cause more than one condition Conditions vary with what is wrong with the gene Some genes can cause disease as AR or AD Conditions vary depending on other genes What will we do when we run out of letters?

Only 6 more LGMDs to go…

CLASSIFICATIONS OF MUSCLE DISEASE

Congenital Myopathy Congenital Muscular Dystrophy

Collagen Related (Bethlem/Ullrich) Dystroglycanopathy Merosin-deficient and related Rigid spine SEPN, Integrins, etc.

Dystrophinopathy EDMD FSHMD Limb Girdle

Dominant, recessive, distal Myasthenia and myasthenic syndromes

EXAMPLE: LMNA Nuclear envelope gene, aka Lamin A/C Emery-Dreifuss muscular dystrophy

Scapuloperoneal, cardiac conduction concerns EDMD 2 and 3, AD (vs. SYNE1, SYNE2, FHL or x-linked with emerin)

LGMD1B AD “laminopathy” CMT2B1 AR

p.R644C missense mutation in the lamin A/C protein Familial partial lipodystrophy FPLD2 AD Dilated cardiomyopathy CMD1A Hutchinson-Gilford progeria

Due to farnesylated mutant prelamin A (progerin)

EXAMPLE: GLYCOSYLATION BUILDING CROSSWALKS

LGMD2I = MDDGC5 = MDC1C (FKRP) LGMD2K = MDDGC1 (POMT1) LGMD2N = MDDGC2 (POMT2) LGMD2O = MDDGC3 (POMGNT1) LGMD2M = MDDGC4 (FKTN)

Japanese severe form of CMD Compare with MDC1a, merosin aka alpha-laminin

LGMD2P = MDDGC9 (DAG1) LGMD2T = MDDGC14 (GMPPB) with/without ID, seizures

We have one case – U of Iowa fibroblast culture rescue technique Walker-Warburg can be several of the above Santavuori (MEB) “cobblestone” usually POMgnT1 “Partial merosin-deficient” likely in this group (MDC1B,D,E or new) CDGS

Classic with transferrin isoelectric focusing Overlap with dystroclycanopathy (DPMs) Brand new (PIGT) – we have two siblings, diagnosed by WES at NIH

GOOD FRIENDS HAVE TO STICK TOGETHER

LGMD & THE JAIN FOUNDATION TOOL

Primarily for LGMD2B, dysferlinopathy Concept is to determine probability and concordance based on

clinical evaluation, FH, CPK, optionally biopsy and brain MRI They got 45/50 if first position, 3/7 if 2 or 3

A route to free 2B or 2I testing Otherwise, great concept but does not quite work yet…

We got 1/12 specific (low concordance on a 2B) We got 4/12 dystroglycanopathy non-specific “definitively excluding conditions based on the presence or absence

of any one symptom isn't a good approach” Working on it, adding Pompe, DYS carrier, etc.

Automated LGMD Diagnostic Assistant (ALDA) http://jain-foundation.org/lgmd-subtyping-tool/content/login-tool

TWO “EASY” CASES

Young girl, brought in as affected DMD female due to biopsy with abnormal dystrophin after high CPK noted Dystrophin gene normal Phenotype merosin-deficient, confirmed genetically

Young man, looked like DMD Dystrophin gene normal, CPK 8,000 Immunostaining with reduced c-terminal dystrophin, COL6,

alpha-dystroglycan, abnormal dysferlin and caveolin pattern

Clinically not Bethlem-Ullrich, CNS near normal Selected LGMD2 gene tests, 2I was positive

TWO “OTHER” CASES

Two girls, one more severely affected Cataracts, club foot, intellectual disability Carrying diagnosis of AMC Genetics conference – consider DM1 vs WES Whole exome indicates CMT 2O (dynamin) Dynein defect reports more similar phenotype

but the two form a complex Mom is low level mosaic Edx (and publication?) pending…

THIS WENT RIGHT TO WES, RIGHTLY

Very severe Duchenne-like presentation 4 year old boy, weak from about 1 year of age,

progressing rapidly CPK only 264, lactate/pyruvate 21./1.6 Biopsy “mixed myopathic” normal

immunostaining WES had defect in thymidine kinase (TK2)

Autosomal recessive Might also have been found in ROH on snp array Mitochondrial DNA depletion, myopathic form

CONCLUSIONS

Single gene test if they walk in out of the textbook, or known diagnosis in family

Non-genetic tests where helpful to narrow it down Only a few of the panel tests make sense – ask Julie! – could it

really be ANY of the genes included? SNP array, mito sequencing may be first step especially if

consanguinity or dysmorphism WES if many possible categories, many possible genes, panels

more expensive NIH seems better than Mayo if we are still stuck (Mayo does not

take AR Medicaid) Genetics, genetic counselors, neuro, pathology, rehab can all

offer clues to differential diagnosis and management – what would happen if we put our heads together?

POST TEST (Easy – T/F)

Neuromuscular diagnosis should usually include a CPK

Neuromuscular diagnosis should always include electrodiagnosis and biopsy