 |
|
Localization of the Gene for Familial Laryngeal Abductor Paralysis to Chromosome 6q16
Jose M. Manaligod, MD;
Jennifer Skaggs, MS;
Richard J. H. Smith, MD
Arch Otolaryngol Head Neck Surg. 2001;127:913-917.
ABSTRACT
| |
Background Vocal fold paralysis is a common cause of neonatal stridor. Although
it is usually classified as idiopathic or iatrogenic in origin, a small subset
of patients have a family history of this disorder, indicating a possible
genetic cause.
Objective To identify the genetic locus of the gene that causes familial laryngeal
abductor paralysis.
Design A standard nonorganic protocol was used to extract DNA from whole-blood
samples. The DNA samples were quantified by DNA fluorometry, and the concentration
of all samples was standardized at 40 ng/µL. A pooled DNA strategy was
used to facilitate rapid polymerase chain reaction screening of markers in
the Weber v8.0 genome screening set. Polymerase chain reaction screening of
individual DNA samples was performed using possible linked markers initially
identified as having an allele that appeared with a higher incidence in the
affected DNA pools. Statistical analysis of possible linkage was performed
using the LINKAGE 5.1 set of linkage analysis computer programs.
Subjects A family in which a form of familial laryngeal abductor paralysis segregates
was ascertained. Whole blood samples were drawn from 40 participating individuals
within this family after the subjects' fully informed consent was obtained.
Results Initial screening of the pooled DNA specimens revealed a band pattern
for D6S1021 on chromosome 6q16, indicating an allele with a higher incidence
in the affected vs the nonaffected pool. Two-point analysis of individual
allele patterns confirmed linkage to D6S1021 with an lod score of 3.86 (
= 0.0) at a penetrance value of 0.8. Haplotype analysis with flanking markers
defined a 5-centiray critical region between D6S283 and AFMA047YG1.
Conclusion An autosomal dominant form of familial laryngeal abductor paralysis
is linked to a 5-centiray region on chromosome 6q16 surrounding D6S1021.
INTRODUCTION
VOCAL FOLD paralysis (VFP) is a frequent cause of congenital stridor
and airway obstruction. Among different laryngeal anomalies causing stridor,
VFP is second only to laryngomalacia in frequency.1
Persistent hoarseness, dysphagia, and recurrent aspiration pneumonia are complications
that may develop because of laryngeal immobility. When significant airway
obstruction exists, it is a potentially life-threatening condition that usually
requires a tracheostomy to provide an adequate airway. Traditionally, VFP
has been classified as acquired (postintubation, injury from cardiothoracic
surgery), neurologic (secondary to Arnold-Chiari malformation), or idiopathic.
However, multigenerational kindreds have been described in which several persons
are afflicted with congenital VFP, indicating a definite genetic component
in these families.
In this study, we used linkage analysis on a large family with an autosomal
dominant form of familial laryngeal abductor paralysis (FLAP) to identify
a genetic locus for this specific form of hereditary vocal fold immobility.
SUBJECTS, MATERIALS, AND METHODS
PATIENTS AND SAMPLES
A family displaying a familial form of FLAP was ascertained through
the University of Iowa Hospitals and Clinics, Iowa City (Figure 1). The study was approved by the institutional review board
of the University of Kentucky, Lexington; appropriate informed consent was
obtained from subjects. Consenting individuals were screened by a directed
history and physical examination, focusing primarily on symptoms of stridor
and cyanosis at birth or persistent hoarseness. Fully affected individuals
also underwent flexible and rigid laryngoscopy and bronchoscopy as part of
their clinical care. The propositus also underwent laryngeal electromyography.
|
|
|
|
Figure 1. Pedigree of a family in which
a form of familial larygeal abductor paralysis segregates with individual
haplotypes. Cross-hatched bars indicate critical intervals that contain familial
laryngeal abductor paralysism loci. Black bars indicate chromosomal regions
excluded by recombination events. Arrow indicates propositus.
|
|
|
GENOTYPING
Following a standard nonorganic protocol, DNA was extracted from 10
mL of whole blood from each consenting individual.2
We used the CHLC (Cooperative Human Linkage Center)/Weber v8.0 human screening
set of short tandem repeat polymorphisms (STRPs) (Research Genetics, Huntsville,
Ala) to complete a genome-wide screen. This screening set contains 386 polymerase
chain reaction (PCR)–based primer pairs that densely cover the entire
human genome with an average intermarker spacing of 10 centimorgans.
To facilitate rapid marker screening, we used a DNA pooling strategy.3-4 Initially, individual samples were
diluted to a concentration of 40 ng/µL. A standard PCR reaction then
was carried out using all individual DNA samples to verify that amplified
STRPs were of equal intensity in all individuals. In cases of unequal amplification,
concentrations were adjusted and the PCR repeated. Once equal DNA concentrations
had been confirmed, affected and nonaffected sample pools were created by
combining equivalent concentrations of the appropriate individual samples.
Rapid PCR screening of the reference panel of markers was carried out
by visually comparing band intensities between the affected and nonaffected
pools. For an autosomal dominant inheritance pattern, 50% of the alleles in
the PCR sample from the affected pool are identical if a STRP is linked to
the disease phenotype. This association means that one band will have a higher
intensity among multiple bands of weaker intensities; in the nonaffected pool,
in contrast, there will be multiple bands of similar intensities.
After possible linked markers were identified, PCR genotyping of individual
samples was performed with the specific STRPs tightly linked to the candidate
marker.5 All PCR products were separated by
electrophoresis on a 6% denaturing acrylamide gel and visualized by autoradiography
through the incorporation of phosphorus 33–labeled dATP (deoxyadenosine
triphosphate) during PCR.
LINKAGE ANALYSIS
Two-point linkage analyses were carried out using the MLINK subroutine
of the LINKAGE package computer programs, version 5.1.6
Maximum lod scores (Zmax) were calculated using the ILINK subroutine of LINKAGE
5.1. Allele frequencies were assumed to be equal for each marker, and recombination
frequencies were assumed to be equal for both sexes. The frequency of the
disease allele was arbitrarily set at 0.0001 because of its rarity.
An assumed penetrance level of 80% was initially used to calculate lod
scores. Additional lod scores for marker D6S1021 were calculated at different
penetrance levels as well, because the actual penetrance of this disorder
is unknown. Multipoint linkage analysis was carried out using the LINKMAP
subroutine of LINKAGE 5.1. Three-point rolling analyses were performed using
adjacent markers and the disease locus, basing marker orders and genetic distances
on the Genethon linkage map (Genethon, Evry France; available at: http://www.genethon.fr).
HAPLOTYPE ANALYSIS
Polymorphic markers that flanked the linked marker by approximately
10 centimorgans were identified using the Human Physical Mapping Project database
of the Center for Genome Research (Whitehead Institute for Biomedical Research,
Cambridge, Mass; available at: http://www.genome.wi.mit.edu/) for
cytogenetic locations of FLAP markers. These markers were used with PCR to
screen the individual DNA samples in order to detect recombination events
centromeric and telomeric to the initial identified marker to define the critical
region that could contain the genetic locus for this disorder.
RESULTS
PATIENTS AND CLINICAL DETAILS
The family we studied displayed a dominantly inherited form of congenital
laryngeal abduction paralysis, as previously described in a report by Manaligod
and Smith.7 In summary, 3 individuals in this
family were identified who required tracheostomy soon after birth for bilateral
vocal fold abductor paralysis. The propositus (individual 40) had absence
of abduction of the vocal folds documented clinically and by laryngeal electromyography.
The other 2 individuals (individuals 26 and 28) were second cousins of the
mother of the propositus. Bilateral laryngeal abductor paralysis was documented
in these 2 individuals by direct laryngoscopy and bronchoscopy prior to tracheostomy.
In all 3 patients, laryngeal adduction appeared intact clinically. No other
neurologic or autonomic abnormalities were evident on physical examination.
All 3 fully affected individuals displayed variable recovery of vocal
fold movement over time. Individual 40 developed partial vocal fold abduction
over the 2 years following his tracheostomy. At age 3 years, although he had
only minimal vocal fold abduction, this patient underwent successful decannulation
of his tracheostomy. Individual 26 showed gradual complete recovery of vocal
fold movement over the first 4 years of life, resulting in decannulation at
age 4 years. Finally, individual 28 recovered only right vocal fold
abduction; nevertheless, she underwent successful decannulation at age 2 years.
After the other family members were questioned, 4 individuals were identified
who had symptoms of stridor and recurrent cyanosis during infancy; these individuals
were classified as affected with decreased expression. In all 4 individuals,
these symptoms gradually resolved over the first 3 to 5 years of life. One
of these individuals, the maternal grandfather of the propositus, underwent
video laryngoscopy, which showed no persistent vocal fold movement abnormalities.
Of the 4 persons meeting these criteria, 2 were obligate carriers. Four other
individuals who were obligate carriers were clinically unaffected. These individuals
were classified as nonpenetrant affected individuals.
GENOTYPING AND LINKAGE ANALYSIS
A whole-genome scan with the Weber v8.0 STRP panel identified only 1
marker, D6S1021 (in the chromosome 6q16 region), as a possible linked marker.
Individual genotyping and linkage analysis (MLINK) performed with this marker
produced a Zmax of 3.86 ( = 0.0) at a penetrance value of 0.8. Additional
genotyping with flanking markers identified a second marker, D6S1546, that
also produced a significant lod score (Zmax = 3.77, = 0.0) (Table 1).
|
|
|
|
Table 1. Pairwise lod Scores for Chromosome 6q16 Markers and Familial
Laryngeal Abductor Paralysis
|
|
|
The Zmax scores for D6S1021 were calculated with penetrance levels between
0.1 and 0.99, and lod scores were significant at all penetrance levels between
0.2 and 0.99 (Table 2). Multipoint
analysis with adjacent markers in the chromosome 6q16 region generated a maximum
lod score of 4.25 between markers D6S1546 and D6S1021 (Figure 2).
|
|
|
|
Table 2. Maximum lod Scores for D6S1021 and Familial Laryngeal Abductor
Paralysis at Varying Penetrance Levels
|
|
|
|
|
|
|
Figure 2. Multipoint analysis of chromosome
6q16 markers and familial laryngeal abductor paralysis. Marker distances (in
parentheses) are in centimorgans (cM) relative to marker D6S1021.
|
|
|
HAPLOTYPE ANALYSIS
To identify the margins of the critical region linked to FLAP, additional
STRP markers within 10 centimorgans of D6S1021 were used for haplotype analysis.
A recombination at marker D6S1692 was noted for individual 28, marking the
centromeric border of the critical region. Another recombination event was
discovered with marker AFMA047YG1, involving individuals 32 and 40, marking
the telomeric border of the region (Figure
1). Based on the Whitehead Institute physical map of chromosome
6, these markers define a 5-centiray region on chromosome 6q16 (Figure 3).
|
|
|
|
Figure 3. Chromosome 6 and putative marker
order in the D6S1021 region.
|
|
|
COMMENT
Vocal fold immobility is a common cause of stridor and airway obstruction
in infants. In a review by Emery and Fearon,8
64% of patients with acquired cases of VFP (intubation or birth trauma) achieved
some degree of spontaneous recovery over time, in contrast to only 29% of
patients with congenital cases (hereditary and idiopathic). In a similarly
structured study, Daya and associates9 saw
a slightly higher rate of recovery of vocal fold motion in idiopathic VFP
(46% [12/26]). An interesting discovery of that report was the recovery of
vocal fold motion in cases of idiopathic bilateral VFP as late as 11 years
of age.
Vocal fold paralysis has been further differentiated based on whether
the primary defect is in vocal fold abduction (glottic opening) or adduction
(glottic closure). Plott10 originally described
the former type of paralysis in a family with 3 brothers who had an X-linked
recessive form of congenital laryngeal abductor paralysis and mental retardation
(Online Mendelian Inheritance in Man; available at: http://www.ncbi.nlm.nih.gov/omim/; Plott syndrome, OMIM 308850). Subsequent reports of families with
both laryngeal paralysis and varying degrees of mental retardation have supported
an X-linked recessive inheritance pattern.11-12
A second syndrome, Gerhardt syndrome, is a form of FLAP with autosomal dominant
inheritance and variable penetrance and expressivity (Gerhardt syndrome, OMIM
150260).13-16
Although mental retardation is not a common feature,7
subtle central neurologic abnormalities can be demonstrated in some kindreds.15
The finding of additional neurologic deficits and other malformations
in conjunction with laryngeal abductor paralysis is common. Pridmore et al17 described a family whose affected members had bilateral
VFP and progressive distal spinal muscular atrophy. In another family, described
by Boltshauser et al,18 affected persons had
similar symptoms in addition to progressive sensorineural hearing loss. Other
known syndromes of central neurologic impairment, such as Möbius syndrome,
occasionally feature laryngeal paralysis if the vagus nerve is affected.19
The most likely anatomic site of involvement in FLAP is the nucleus
ambiguus.16 Genetic abnormalities that affect
the development of this central nucleus also may affect those portions of
the brain and brainstem that develop concomitantly. The result would be phenotypic
heterogeneity and variability in the severity of the laryngeal paralysis.
In this study, we demonstrated statistically significant linkage between
D6S1021 and the form of FLAP segregating in this family. This is the first
definitively identified locus for any form of laryngeal dysfunction. Haplotype
analysis narrows the critical region to a 5-centiray interval on chromosome
6q16 flanked by D6S1692 and AFMA047YG1. Twenty-four uncharacterized expressed
sequence tags and several genes have been mapped to this area (GeneMap '99;
National Center for Biotechnology Information, Bethesda, Md/International
Radiation Hybrid Mapping Consortium) and to several genes, including KIAA0331, PREP (prolyl endopeptidase,
an endopeptidase that cleaves neuropeptides), FKHRL1
(forkhead Drosophila homolog rhabdomyosarcoma like
1), CDW52, ASP (apoptosis-specific
protein, a protein specific for programmed cell death) sequences, and GPR6 (G protein–coupled receptor 6). Both PREP and ASP may have roles in neurological
and developmental function and should be considered as particularly strong
candidate genes for FLAP.
Future efforts to identify the specific gene mutation that causes this
disorder should focus primarily on genes expressed in the central nervous
system that show sequence homology to known genes implicated in neural development
and differentiation. Because this family (as well as other described pedigrees)
displays anticipation, a phenomenon caused by expansile trinucleotide repeats
in diseases such as fragile X syndrome and Friedreich ataxia, the same process
may underlie this disorder. Therefore, candidate genes within this interval
that contain trinucleotide repeats should be considered strong possibilities
for the FLAP gene at this locus.
AUTHOR INFORMATION
Accepted for publication February 7, 2001.
This research was enabled by support from the Kentucky Children's Miracle
Network, Lexington.
We are extremely grateful to the family members who participated in
this study.
Corresponding author and reprints: Jose M. Manaligod, MD, Department
of Otolaryngology–Head and Neck Surgery, University of Iowa Hospitals
and Clinics, 200 Hawkins Dr, Iowa City, IA 52242 (e-mail:
jose-manaligod{at}uiowa.edu).
From the University of Kentucky Medical Center, Lexington (Dr Manaligod
and Ms Skaggs); and the University of Iowa Hospitals and Clinics, Iowa City
(Dr Smith). Dr Manaligod is now with the Department of Otolaryngology–Head
and Neck Surgery, University of Iowa Hospitals and Clinics.
REFERENCES
| |
1. Zbar RI, Smith RJ. Vocal fold paralysis in infants twelve months of age and younger. Otolaryngol Head Neck Surg. 1996;114:18-21.
FULL TEXT
|
ISI
| PUBMED
2. Grimberg J, Nawoschik S, Belluscio L, McKee R, Turck A, Eisenberg A. A simple and efficient non-organic procedure for the isolation of genomic
DNA from blood. Nucleic Acids Res. 1989;17:8390.
FREE FULL TEXT
3. Sheffield VC, Nishimura DY, Stone EM. Novel approaches to linkage mapping [review]. Curr Opin Genet Dev. 1995;5:335-341.
FULL TEXT
|
ISI
| PUBMED
4. Sheffield VC, Pierpont ME, Nishimura D, et al. Identification of a complex congenital heart defect susceptibility
locus by using DNA pooling and shared segment analysis. Hum Mol Genet. 1997;6:117-121.
FREE FULL TEXT
5. Chen A, Mueller R, Prasad S, et al. Presymptomatic diagnosis of nonsyndromic hearing loss by genotyping
[review]. Arch Otolaryngol Head Neck Surg. 1998;124:20-24.
FREE FULL TEXT
6. Lathrop GM, Lalouel JM, Julier C, Ott J. Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci U S A. 1984;81:3443-3446.
FREE FULL TEXT
7. Manaligod JM, Smith RJ. Familial laryngeal paralysis. Am J Med Genet. 1998;77:277-280.
FULL TEXT
|
ISI
| PUBMED
8. Emery P, Fearon B. Vocal cord palsy in pediatric practice: a review of 71 cases. Int J Pediatr Otorhinolaryngol. 1984;8:147-154.
FULL TEXT
|
ISI
| PUBMED
9. Daya H, Hosni A, Bejar-Solar I, Evans JNG, Bailey CM. Pediatric vocal fold paralysis: a long-term retrospective study. Arch Otolaryngol Head Neck Surg. 2000;126:21-25.
FREE FULL TEXT
10. Plott D. Congenital laryngeal-abductor paralysis due to nucleus ambiguus dysgenesis
in three brothers. N Engl J Med. 1964;271:593-597.
11. Opitz JM, Kaveggia E, Durkin-Stamm M, Pendelton E. Diagnostic/genetic studies in severe mental retardation. Birth Defects Orig Artic Ser. 1978;14:1-38.
12. Watters G, Fitch N. Familial laryngeal abductor paralysis and psychomotor retardation. Clin Genet. 1973;4:429-433.
ISI
| PUBMED
13. Cunningham MJ, Eavey RD, Shannon DC. Familial vocal cord dysfunction. Pediatrics. 1985;76:750-753.
FREE FULL TEXT
14. Gacek RR. Hereditary abductor vocal cord paralysis. Ann Otol Rhinol Laryngol. 1976;85:90-93.
15. Grundfast KM, Milmoe G. Congenital hereditary bilateral abductor vocal cord paralysis. Ann Otol Rhinol Laryngol. 1982;91(6, pt 1):564-566.
16. Morelli G, Mesolella C, Costa F, Testa B, Ventruto V, Santulli S. Familial laryngeal abductor paralysis with presumed autosomal dominant
inheritance. Ann Otol Rhinol Laryngol. 1982;91(3 pt 1):323-324.
17. Pridmore C, Baraitser M, Brett EM, Harding AE. Distal spinal muscular atrophy with vocal cord paralysis. J Med Genet. 1992;29:197-199.
FREE FULL TEXT
18. Boltshauser E, Lang W, Spillmann T, Hof E. Hereditary distal muscular atrophy with vocal cord paralysis and sensorineural
hearing loss: a dominant form of spinal muscular atrophy. J Med Genet. 1989;26:105-108.
FREE FULL TEXT
19. Cohen SR, Thompson JW. Variants of Möbius' syndrome and central neurologic impairment:
Lindeman procedure in children [review]. Ann Otol Rhinol Laryngol. 1987;96:93-100.
ISI
| PUBMED
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati
What's this?
RELATED ARTICLE
Archives of Otolaryngology–Head & Neck Surgery Reader's Choice: Continuing Medical Education
Arch Otolaryngol Head Neck Surg. 2001;127(8):1011-1012.
FULL TEXT
|
