FHL1-Myopathies via the FHL1 Gene
- Summary and Pricing
- Clinical Features and Genetics
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The great majority of tests are completed within 18 days.
Analytical sensitivity should be high because nearly all FHL1 mutations reported to date are detectable by direct sequencing of genomic DNA.
Disorders of skeletal muscle resulting from mutations in the FHL1 gene vary by age of onset, rate of progression, severity of clinical presentation, and findings from muscle histopathology. Severe, early onset reducing body myopathy (OMIM 300717) is characterized by early onset and progressive hypotonia, proximal muscle weakness, contractures, and respiratory insufficiency. Patients are normal at birth until they develop proximal weakness as toddlers (Kiyomoto et al. J Neurol Sci 128:58-65, 1995; Schessl et al. Brain 132:452-464, 2009). In the reported patients, disease progression was rapid and respiratory failure occurred 2-3 years after disease onset. Initial clinical signs included frequent falls, abnormal gait, progressive proximal muscle weakness, contractures, and scoliosis. Childhood onset reducing body myopathy (OMIM 300718) exhibits onset between 5 and 16 years of age. Disease progression is also rapid with patients becoming wheelchair bound one and three years from the time of onset (Goebel et al. Neuropediatrics 32:196-205, 2001; Schessl et al. J Clin Invest 118:904-912, 2008). Clinical features include proximal weakness, joint contractures, spinal rigidity, scoliosis, scapular winging, Gowers sign, and decreased deep tendon reflexes (Ohsawa et al. Brain Dev 29:112-116, 2007, Liewluck et al. Muscle Nerve 35:322-326, 2007). Mothers of affected boys have milder symptoms later in life compared to their children.
X linked myopathy with postural muscle atrophy (OMIM 300696) is an adult onset disorder with weakness and atrophy of muscles required for postural stability. Patients often exhibit muscle hypertrophy early in life and in non proximal muscle groups. Among a group of seven unrelated families, disease onset was between 8 and 45 years of age and women carriers were either asymptomatic or mildly affected (Schoser et al. Neurology 73:543-551, 2009). Patients in this study had early-onset neck rigidity, contractures of the Achilles tendon, progressive limb girdle muscle weakness, postural muscle atrophy, rigid spine, scapular winging, gait abnormalities, and respiratory insufficiency.
Emery-Dreifuss muscular dystrophy-6 (OMIM 300696) is a closely related clinical phenotype to that of X linked myopathy with postural muscle atrophy. Age at onset has been found to be in the first to second decades of life in two studies (Gueneau et al. Am J Hum Genet 85:338-353, 2009; Knoblauch et al. Ann Neurol 67:136-140, 2010). Patients develop cardiac muscle involvement, including arrhythmias and hypertrophic cardiomyopathy, after onset of skeletal muscle symptoms. Clinical features include joint contractures, neck stiffness, and limb girdle muscle weakness and atrophy.
All patients reported have remained ambulatory. Female carriers are rarely affected (Gueneau et al. Am J Hum Genet 85: 338-353, 2009). Scapuloperoneal myopathy (OMIM 300695) has been described in a single family (Wilhelmsen et al. Ann Neurol 39:507-520, 1996). Clinical features include foot drop, proximal arm weakness which precedes hand weakness, and scapular winging. Affected men showed signs earlier in life than women and were more severely affected (Quinzii et al. Am J Hum Genet 82:208-213, 2008).
Patients with myofibrilar myopathy with reducing bodies seen in muscle biopsies have also been found with FHL1 gene mutations (Selcen et al. Neurology 77:1951, 2011).
Reducing bodies are irregularly shaped cytoplasmic inclusions that appear dark with menadione-NBT stain, confirming the presence of sulfhydryl groups. FHL1 protein, both mutant and normal forms, is the most abundant protein found in reducing bodies (Schessl et al. J Clin Invest 118:904–912, 2008; Schessl et al. Brain 132:452-464, 2009). Muscle biopsies of patients with FHL1 myopathies reveal variation in fiber size, internally placed nuclei, mild inflammation, rimmed vacuoles, and reducing bodies that stain with NBT.
FHL1-related myopathies are inherited as X-linked recessive disorders or, in the case of scapuloperoneal myopathy, as an X-linked dominant disorder. In familial cases, mothers of affected males are themselves clinically less affected with symptoms appearing later in life. Severe, early onset reducing body myopathy results from de novo missense mutation of conserved amino acids of the second LIM domain (Schessl et al. J Clin Invest 118:904-912, 2008; Shalaby et al. Neurology 72:375-376, 2009). Mutations involving the zinc coordinating residue p.His123 result in a severe clinical course, while mutations in another zinc coordinating residue (p.Cys153) are associated with a milder phenotype. Childhood onset reducing body myopathy results from inherited mutations of the second LIM domain. The scapuloperoneal myopathy mutation is found in the second LIM domain and affects a conserved residue (p.Trp122Ser). Patients with Emery-Dreifuss muscular dystrophy-6 more often have FHL1 mutations in the fourth LIM domain. The majority of reported mutations in FHL1 are missense or nonsense (Human Gene Mutation Database).
The ‘four-and-a-half LIM domain 1’ protein is coded by exons 3-8 of the FHL1 gene (OMIM 300163) on chromosome Xq26.3. Testing is accomplished by amplifying each coding exon and ~20 bp of adjacent noncoding sequence, then determining the nucleotide sequence using standard dideoxy Sanger sequencing methods and a capillary electrophoresis instrument. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results.
Indications for Test
Patients with clinical and histopathological features of FHL1-related disorders.
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- Genetic Counselor Team - firstname.lastname@example.org
- Angela Gruber, PhD - email@example.com
- Goebel, H. H., et al. (2001) Reducing body myopathy with cytoplasmic bodies and rigid spine syndrome: a mixed congenital myopathy. Neuropediatrics 32: 196-205. PubMed ID: 11571700
- Gueneau, L., et al. (2009) Mutations of the FHL1 gene cause Emery-Dreifuss muscular dystrophy. Am. J. Hum. Genet. 85: 338-353. PubMed ID: 19716112
- Human Gene Mutation Database.
- Kiyomoto, B. H., et al. (1995) Fatal reducing body myopathy: ultrastructural and immnunohistochemical (sic) observations. J. Neurol. Sci. 128: 58-65. PubMed ID: 7722535
- Knoblauch, H. et al. (2010) Contractures and hypertrophic cardiomyopathy in a novel FHL1 mutation. Ann. Neurol. 67: 136-140. PubMed ID: 20186852
- Liewluck, T. et al. (2007) Unfolded protein response and aggresome formation in hereditary reducing-body myopathy. Muscle Nerve 35: 322-326. PubMed ID: 17099882
- Ohsawa, M. et al. (2007) Familial reducing body myopathy. Brain Dev. 29: 112-116. PubMed ID: 16919903
- Quinzii, C. M. et al. (2008) X-linked dominant scapuloperoneal myopathy is due to mutation in the gene encoding four-and-a-half-LIM protein 1. Am. J. Hum. Genet. 82: 208-213. PubMed ID: 18179901
- Schessl, J. et al. (2008). Proteomic identification of FHL1 as the protein mutated in human reducing body myopathy. J. Clin. Invest. 118: 904-912. PubMed ID: 18274675
- Schessl, J. et al. (2009). Clinical, histological and genetic characterization of reducing body myopathy caused by mutations in FHL1. Brain 132: 452-464. PubMed ID: 19181672
- Schoser, B. et al. (2009) Consequences of mutations within the C terminus of the FHL1 gene. Neurology 73: 543-551. PubMed ID: 19687455
- Selcen, D. et al. (2011) Reducing bodies and myofibrillar myopathy features in FHL1 muscular dystrophy. Neurology 77:1951-1959. PubMed ID: 22094483)
- Shalaby, S. et al. 2009. Novel FHL1 mutations in fatal and benign reducing body myopathy. Neurology 72(4):375-6. PubMed ID: 19171836
- Wilhelmsen, K. C. et al. (1996) Chromosome 12-linked autosomal dominant scapuloperoneal muscular dystrophy. Ann. Neurol. 39: 507-520. PubMed ID: 8619529
Bi-Directional Sanger Sequencing
Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org). As required, DNA is extracted from the patient specimen. PCR is used to amplify the indicated exons plus additional flanking non-coding sequence. After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions. In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.
As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.
Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).
In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.
Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.
In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.
In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.
Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.
Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.
Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.
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(Delivery accepted Monday - Saturday)
- Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
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- Only one blood tube is required for multiple tests.
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- During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
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- At room temperature, blood specimen is stable for up to 48 hours.
- If refrigerated, blood specimen is stable for up to one week.
- Label the tube with the patient name, date of birth and/or ID number.
(Delivery accepted Monday - Saturday)
- Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
- For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
- DNA may be shipped at room temperature.
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- Culture and send at least two T25 flasks of confluent cells.
- Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
- Send specimens in insulated, shatterproof container overnight.
- Cell cultures may be shipped at room temperature or refrigerated.
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- We strongly recommend maintaining a local back-up culture. We do not culture cells.