Primary ciliary dyskinesia (PCD) is an autosomal recessive multigenic disease that results in impaired mucociliary clearance. We have diagnosed 9 subjects with primary ciliary dyskinesia from geographically dispersed Amish communities, based on clinical characteristics and ciliary ultrastructural defects. Despite consanguinity, affected individuals had evidence of genetic heterogeneity

Bowcock, Anne M.

Brown, David E.

Carson, John L.

Ferkol, Thomas W.

Forsen, James

Hazucha, Milan J.

Helms, Cynthia

Knowles, Michael R.

Leigh, Margaret W.

Lie, Hauw

Molter, David

Zariwala, Maimoona A.

PubMed

Carolina Digital Repository

Primary ciliary dyskinesia in Amish communitiesHauw Lie1, Maimoona A Zariwala5, Cynthia Helms2, Anne M Bowcock2, John L Carson4,David E Brown III4, Milan J Hazucha5, James Forsen3, David Molter3, Michael R Knowles6,Margaret W Leigh4, and Thomas W Ferkol11Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri2Department of Genetics, Washington University School of Medicine, St. Louis, Missouri3Department of Otolaryngology, Washington University School of Medicine, St. Louis, Missouri4Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina5Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NorthCarolina6Department of Medicine, University of North Carolina, Chapel Hill, North CarolinaAbstractPrimary ciliary dyskinesia (PCD) is an autosomal recessive multigenic disease that results in impairedmucociliary clearance. We have diagnosed 9 subjects with primary ciliary dyskinesia fromgeographically dispersed Amish communities, based on clinical characteristics and ciliaryultrastructural defects. Despite consanguinity, affected individuals had evidence of geneticheterogeneity.KeywordsPrimary ciliary dyskinesia; cilia; dynein; nitric oxide; AmishPrimary ciliary dyskinesia (PCD) is an inherited disorder characterized by functionalimpairment of ciliary motility, clinically manifested as chronic bronchitis, pansinusitis, andsuppurative otitis media (1). Evidence of ciliary dysfunction can occur as early as birth withrespiratory distress in the immediate newborn period (2). Ciliary dysfunction can lead tolaterality defects, e.g. situs inversus totalis (3,4). As predicted from the complexity of ciliarystructure and function, PCD is genetically heterogeneous, involving multiple genes. In mostinstances, the disease is expressed in an autosomal recessive patternFirst settling in rural Pennsylvania, close-knit agrarian Amish communities have spread acrossNorth America. Because members rarely marry outside the church, consanguinity is commonresulting in closed populations with a high frequency of genetic disorders. Intermarriage,© 2009 Mosby, Inc. All rights reserved.Corresponding author: Thomas Ferkol, MD, Division of Pediatric Allergy, Immunology, and Pulmonary Medicine, Department ofPediatrics, 660 South Euclid Avenue, Mailbox 8116, St. Louis, Missouri, 63110. Telephone: 314 286 2886. Facsimile: 314 454 2515.ferkol_t@kids.wustl.edu.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.The authors declare no conflicts of interest.NIH Public AccessAuthor ManuscriptJ Pediatr. Author manuscript; available in PMC 2011 June 1.Published in final edited form as:J Pediatr. 2010 June ; 156(6): 1023–1025. doi:10.1016/j.jpeds.2010.01.054.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptrelative isolation, and detailed record-keeping make Amish communities ideal for geneticstudies. We found that several children in geographically dispersed Amish communities hadPCD, and hypothesized that additional evaluation would confirm a high frequency of PCD inthese communities and identify a founder mutation.METHODS23 individuals from Amish communities in the Midwest who had respiratory distress as terminfants in the immediate neonatal period, situs inversus totalis, recurrent pneumonia, chronicsinusitis, or male infertility were recruited at a one-day outreach clinic for further study. Signedinformed consent was obtained from all subjects and family members.Evaluations included detailed clinical and family histories, physical examination, andotolaryngological assessment. Nasal nitric oxide (NO) levels were measured using establishedtechniques (model CLD 88, Eco Medics AG). Spirometry and audiometry were performed onsubjects 5 years or older. Sputum or oropharyngeal swabs were collected for microbiologicalstudies. Nasal scrapings from inferior nasal turbinates were obtained to collect ciliatedepithelial cells for ultrastructural analysis. Blood or buccal cell samples were collected forgenetic analysis; DNA was extracted using a commercially available DNA extraction kit.Whole genome amplification (REPLI-g kit, Qiagen) was performed on each sample to provideadequate genetic material for testing. Focused, mutational analysis and sequencing ofDNAI1 was performed. Allele image profiles were generated using genotyping derived from10K single nucleotide polymorphism arrays (Affimetrix) and microsatellite markers, shortDNA segments with repeated, specific motifs 1–6 bases long, for candidate genes (DNAI1,DNAH5, DNAH11, DNAH7, DNAH9, DNAI2, DNAL4, DNAH3, and TCTEL1). Haplotypeswere reconstructed such that genotypes could be visualized on pedigrees, and homozygositymapping was used to identify a common genetic locus in the consanguineous family.RESULTS15 males and 8 females from infancy to adulthood (age range 3 months to 45 years) underwentevaluation. Based on ciliary ultrastructural analysis, 9 of the subjects (6 male) evaluated wereconfirmed to have dynein arm defects (absence of outer dynein arm) consistent with PCD.Subjects with PCD ranged in age from 3 months to 40 years (median age 14 years).All were born at term, and 8 cases had neonatal respiratory distress (8/9). Every affectedindividual had persistent nasal congestion and coughing. Other clinical findings includedlaterality defects (5/9), chronic rhinosinusitis (8/9), and pneumonia (5/9). Surprisingly, onlyone subject (1/9) had a history of recurrent, symptomatic acute otitis media. However,audiological evaluations performed on 5 older children and adults revealed mildly ormoderately abnormal tympanometry measurements and conductive hearing loss thought to bedue to otitis media with effusion.In the 4 subjects with PCD who could perform spirometry, the mean FVC was 74.5% (range55 to 82%) and mean FEV1 was 66.5% predicted for age (range 36 to 80%). Bacterial pathogensisolated from sputum cultures included Haemophilus influenzae (6/9), Streptococcuspneumoniae (2/9), and Pseudomonas aeruginosa (1/9). Atypical mycobacteria were isolatedfrom an adult PCD subject (1/9).Nasal NO measurements were extremely low in these subjects ranging from 1.2 to 11.1 nL/min with all 9 PCD subjects having NO levels < 14 nL/min.Initial genetic testing from those with confirmed PCD at selected exons identified 3 individualscarrying a founder splice mutation (IVS1+2_3insT) of DNAI1, the most common mutationLie et al. Page 2J Pediatr. Author manuscript; available in PMC 2011 June 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptassociated with PCD (Figure). This mutation was present on a single allele, and full sequencingof DNAI1 in these subjects did not reveal a second mutation. Genotype analysis of selectedmicrosatellites from candidate genes followed Mendelian patterns of inheritance, based onhaplotypes. No concordance was found in affected individuals within the same family withany microsatellite marker tested, indicating that affected individuals harbor a disease-causingmutation in a previously unidentified PCD gene. Genome-wide linkage search of closelyrelated PCD subjects was performed, and multipoint linkage analysis identified 4 regions ofhigh concordance on chromosomes 2p, 4p, 6p, and 13p.DISCUSSIONWe describe members from related Amish communities who have PCD, based on clinicalevaluation and ultrastructural defects of the ciliary axoneme. Extensive, genetic analyses,however, indicated significant genetic heterogeneity.PCD has been estimated to occur in approximately 1 in 12,000 to 20,000 live births (3,4). Thecurrent “gold standard” for diagnosis relies on identification of ciliary ultrastructuralabnormalities by transmission electron microscopy (5), but this method has drawbacks (6).Fuller understanding of the genetic basis of PCD may lead to another, superior diagnosticmodality to identify affected individuals. The genetic heterogeneity of PCD is predicted by thecomplexity of the ciliary structure (7). Several potential candidate dynein genes are involvedin PCD, including mutations in DNAI1 (8) and DNAH5 (9). Other dynein arm-coding genes,DNAH11 and DNAH7, have also been associated with PCD (10).All subjects in this population with confirmed PCD reported chronic nasal congestion andcough. Although there are other causes of neonatal respiratory distress, our population supportsthe observation that neonatal respiratory distress is a common presenting feature of PCD.Surprisingly, only one subject had a history of chronic middle ear involvement, a featurethought to be common to all PCD patients. The reason for the low prevalence of otitis mediain this population is unclear, though it is likely related in part to underreporting.Spirometry revealed significant intrathoracic airway obstruction in subjects who could performpulmonary function testing, though it is difficult to make conclusions based on the few subjectsstudied. Nasal NO concentrations were low in all subjects with PCD, confirming previousobservations that individuals with PCD typically have reduced nasal NO production (10).In conclusion, we have found that several members of related, geographically dispersed, Amishfamilies have PCD. Although the specific genetic causes for most PCD are still unknown, suchfamilies provide us with an opportunity to understand this “rare” lung disease. We havecollected extensive clinical and family data describing affected individuals, which should allowus to better define the genetics of disease and phenotype-genotype relationships. Already, wehave found unexpected genetic heterogeneity in this inbred Amish community. Furtherevaluation is necessary to identify the specific mutations in this population.AcknowledgmentsThe authors would like to thank Jane Quante, Susan Minnix, and Kimberly Burns for their expert assistance on theproject. We also appreciate the efforts of Katherine Rivera, Lila Kertz, Kristina Shilts, Carolyn Cannon, John Spivey,and Deborah White, who participated in the outreach clinic.Supported by National Institutes of Health awards RR019480 (M.R.K.), HL071798 (M.R.K.), and HL079024(T.W.F.), and the Children’s Discovery Institute (T.W.F.).Lie et al. Page 3J Pediatr. Author manuscript; available in PMC 2011 June 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptABBREVIATIONSNO nitric oxidePCD primary ciliary dyskinesiaDNAI1 dynein axonemal intermediate chain 1REFERENCES1. Knowles MR, Boucher RC. Mucus clearance as a primary innate defense mechanism for mammalianairways. J Clin Invest 2002;209:571–577. [PubMed: 11877463]2. Noone PG, Leigh MW, Sannuti A, Minnix SL, Carson JL, Hazucha M, Zariwala MA, Knowles MR.Primary ciliary dyskinesia: diagnostic and phenotypic features. Am J Respir Crit Care Med2004;169:459–467. [PubMed: 14656747]3. Torgersen J. Situs inversus, asymmetry, and twinning. Am J Hum Genet 1950;2:361–370. [PubMed:14837905]4. Katsuhara K, Kawamoto S, Wakahayashi T, et al. Situs inversus totalis and Kartagener’s syndrome ina Japanese population. Chest 1972;61:56–61. [PubMed: 4538074]5. Bush A, Chodhari R, Collins N, Copeland F, Hall P, Harcourt J, Hariri M, Hogg C, Lucas J, MitchisonHM, O'Callaghan C, Phillips G. Primary ciliary dyskinesia: current state of the art. Arch Dis Child2007;92:1136–1140. [PubMed: 17634184]6. Ferkol T, Leigh M. Primary ciliary dyskinesia and newborn respiratory distress. Semin Perinatol2006;30:335–340. [PubMed: 17142159]7. Dutcher SK. Flagellar assembly in two hundred and fifty easy-to-follow steps. Trends Genet1995;11:398–404. [PubMed: 7482766]8. Zariwala MA, Leigh MW, Ceppa F, Kennedy MP, Noone PG, Carson JL, Hazucha MJ, Lori A, HorvathJ, Olbrich H, Loges NT, Bridoux AM, Pennarun G, Duriez B, Escudier E, Mitchison HM, ChodhariR, Chung EM, Morgan LC, de Iongh RU, Rutland J, Pradal U, Omran H, Amselem S, Knowles MR.Mutations of DNAI1 in primary ciliary dyskinesia: evidence of founder effect in a common mutation.Am J Respir Crit Care Med 2006;174:858–866. [PubMed: 16858015]9. Hornef N, Olbrich H, Horvath J, Zariwala MA, Fliegauf M, Loges NT, Wildhaber J, Noone PG,Kennedy M, Antonarakis SE, Blouin JL, Bartoloni L, Nusslein T, Ahrens P, Griese M, Kuhl H, SudbrakR, Knowles MR, Reinhardt R, Omran H. DNAH5 mutations are a common cause of primary ciliarydyskinesia with outer dynein arm defects. Am J Respir Crit Care Med 2006;174:120–126. [PubMed:16627867]10. Leigh MW, Pittman JE, Carson JL, Ferkol TW, Dell SD, Davis SD, Knowles MR, Zariwala MA.Clinical and genetic aspects of primary ciliary dyskinesia/Kartagener syndrome. Genet Med2009;11:473–487. [PubMed: 19606528]Lie et al. Page 4J Pediatr. Author manuscript; available in PMC 2011 June 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure.Pedigree of consanguineous kindred with related nuclear families. Legend: si, situs inversus;sa, situs ambiguus; ODA, outer dynein arm defect; DNAI1, dynein axonemal intermediatechain 1; +/−, heterozygous. Symbols: □ male, ○ female, and filled symbols indicate affectedindividuals. Three subjects with confirmed PCD carried a splice mutation (IVS1+2_3insT) ofDNAI1, and the other 6 affected individuals were negative for this founder mutation.Lie et al. Page 5J Pediatr. Author manuscript; available in PMC 2011 June 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

Primary Ciliary Dyskinesia in Amish Communities

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Primary Ciliary Dyskinesia in Amish Communities

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