Primary Ciliary Dyskinesia Next-Gen Sequencing Panel (includes CFTR)

Primary Ciliary Dyskinesia (PCD), also called Immotile Cilia
Syndrome (ICS), is a genetically heterogeneous, usually autosomal recessive disorder with impaired ciliary function leading to progressive sinopulmonary disease.

Upper and lower respiratory tract manifestations are key features of PCD and are often present at birth. Upper respiratory tract features include chronic nasal drainage, sinusitis, and otitis media. Lower respiratory features include neonatal respiratory distress, chronic productive cough, chronic bronchitis, recurrent pneumonia, and bronchiectasis. Approximately half of PCD patients have situs inversus, and the triad of sinusitis, bronchiectasis and situs inversus is also known as Kartagener’s syndrome. Approximately ~6% of individuals with PCD have situs ambiguous (heterotaxy), which is attributed to dysmobility of embryological nodal cilia. The prevalence of congenital heart disease is 200 fold higher in PCD that in the general population. In adults with PCD, male infertility and female sub-fertility are also common. Prompt diagnosis of PCD is critical for the prevention of secondary respiratory complications.

Identification of PCD and PCD-Related Disorders gene mutations can aid diagnosis and may dramatically improve outcome. Comprehensive testing is indicated in patients who are clinically suspected of having PCD and PCD-Related Disorders. The PCD and PCD-Related Disorders Next-Gen Sequencing Panel may also be considered for differential diagnosis, for carrier testing in individuals with a family history, and for at-risk pregnancies.

The Ambry Test: PCD Next-Gen Sequencing Panel detects mutations in all coding domains and splice junctions of the DNAAF1/ LRRC50, DNAAF2/ c14orf104, DNAH5, DNAH11, DNAI1, DNAI2, RSPH4A, RSPH9, TXNDC3, OFD1, RPGR and CFTR genes. This Ambry test identifies >92.6% of described PCD mutations (analytic sensitivity). Approximately 56% of patients with PCD and PCD-Related Disorders have a detectable mutation in the listed sequencing panel genes (clinical sensitivity).

Disease Name

Primary Ciliary Dyskinesia (PCD)

Immotile Cilia Syndrome (ICS)

Kartagener's Syndrome

Disease Information

Primary Ciliary Dyskinesia (PCD), also called Immotile Cilia Syndrome (ICS), is a genetically heterogeneous, usually autosomal recessive disorder with impaired ciliary function leading to progressive sinopulmonary disease.^1,2 Upper and lower respiratory tract manifestations are key features of PCD and are often present at birth. Upper respiratory tract features include chronic nasal drainage, sinusitis, and otitis media. Lower respiratory features include neonatal respiratory distress, chronic productive cough, chronic bronchitis, recurrent pneumonia, and bronchiectasis.^1,3 Approximately half of the PCD patients have situs inversus, and the triad of sinusitis, bronchiectasis and situs inversus is also known asKartagener’s syndrome.^1,2

At least ~6% of individuals with PCD have situs ambiguous (heterotaxy), which is attributed to dysmobility of embryological nodal cilia.^3,4 The prevalence of congenital heart disease is 200 fold higher in PCD that in the general population.⁴In adults with PCD, male infertility and female sub-fertility are also common.^2,3 Prompt diagnosis of PCD is critical for the prevention of secondary respiratory complications. The incidence of PCD estimated to be 1:16000 worldwide² and 1:12,000 to 1:17,000 in the United States.³ Defective dynein arms occur in ~90% of individuals who have defined ultrastructural defects; and ~10% have defective central pair or radial spoke or nexin links.

The genes in which disease-causing mutations have been identified include nine autosomal genes coding for outer dynein arm (ODA) proteins: (DNAI1, DNAI2, DNAH5, DNAH11, TXNDC3),^5-9 radial spoke (RS) proteins (RSPH4A, RSPH9),¹⁰ and cytoplasmic proteins involved in dynein arm (DA) assembly (DNAAF1/LRRC50, DNAAF2/c14orf104/KTU)^11-12. Two X-linked genes have been implicated in PCD. Mutations in RPGR¹³ have been identified in patients with retinitis pigmentosa and PCD 11 and OFD1 mutations have been identified in patients with mental retardation, macrocephaly and PCD¹⁴. The three additional autosomal genes that were more recently implicated in PCD but are not included on this panel are CCDC39¹, CCDC40², and DNAL1³. Due to the relative high incidence of cystic fibrosis (CF), and the phenotypic overlap between PCD and CF, the CFTR gene testing was included to address a potential genetic diagnosis of cystic fibrosis.

Diagnosis of PCD has relied on identification of ciliary dysmotility and specific ciliary ultrastructural defects, but ultrastructural studies are challenging and not readily available. Establishing the cause of PCD is essential for prognosis, management, and genetic counseling. Mutations in DNAI1 and DNAH5 are identified in an estimated 38% of all PCD patients, and in as many as 60% of PCD patients with defects in ciliary ODA.^5,15 With all 11 PCD genes, clinical detection rates are estimated to be approximately 56-59.6% using this panel.

Testing Benefits & Indication

Identification of PCD and PCD-related disorders gene mutations can aid diagnosis and may dramatically improve outcome. Comprehensive testing is indicated in patients who are clinically suspected to PCD and PCD-related disorders. The PCD Next-Gen Sequencing Panel may also be considered for differential diagnosis, carrier testing for individuals with a family history, and for at-risk pregnancies.

Indications for DNA testing for PCD and PCD-related disorders include:

Known or suspected primary ciliary dyskinesia
Chronic sinusitis or bronchiectasis
Suspected PCD with heterotaxy, situs ambiguus, or situs inversus (Kartagener Syndrome)
Congenital heart defect associated with recurrent respiratory disease or heterotaxy
Chronic otitis media with effusion
Male infertility with other signs of PCD
Idiopathic respiratory distress in full-term neonates
Carrier status determination for relatives of patients with known mutations

Test Description

The PCD Next-Gen Sequencing Panel is a comprehensive gene sequencing screen for genes associated with PCD and PCD-related disorders. Genomic deoxyribonucleic acid (gDNA) is isolated from the patient’s specimen using a standardized kit and quantified by agarose gel electrophoresis. Sequence enrichment is carried out by incorporating the gDNA into microdroplets along with primer pairs designed to the target gene coding exons followed by polymerase chain reaction (PCR) and Next-Generation sequencing.

Additional Sanger sequencing is performed for any regions missing or with insufficient read depth coverage for reliable heterozygous variant detection. Suspect variant calls other than polymorphisms are verified by Sanger sequencing in sense and antisense directions.The PCD NextGen Sequencing Panel is a comprehensive gene sequencing screen for genes associated with PCD and PCD-related disorders. Genomic deoxyribonucleic acid (gDNA) is isolated from the patient’s specimen using a standardized kit and quantified by agarose gel electrophoresis. Sequence enrichment is carried out by incorporating the gDNA into microdroplets along with primer pairs designed to the target gene coding exons followed by polymerase chain reaction (PCR) and Next-Generation sequencing.

Additional Sanger sequencing is performed for any regions missing or with insufficient read depth coverage for reliable heterozygous variant detection. Suspect variant calls other than polymorphisms are verified by Sanger sequencing in sense and antisense directions.

The PCD Next-Gen Sequencing Panel detects mutations in all coding domains and splice junctions DNAAF1/ LRRC50, DNAAF2/ c14orf104, DNAH5, DNAH11, DNAI1, DNAI2, RSPH4A, RSPH9, TXNDC3, OFD1, RPGR and CFTR genes.

12 Genes Represented on the PCD and PCD-Related Disorders Next-Gen Sequencing Panel

Gene	OMIM #	PCD Form	Full Gene Name	Chr	Disease	Accession Number	cDNA (bp) and aa	PCD Clinical Sensitivity
*LRRC50 (DNAAF1)*	*613190	PCD 13	Dynein, axonemal, assembly factor 1	16	PCD, KS	NM_178452.4	2178 (725aa)	5% ^{Ref 9}
*c14orf104/KTU (DNAAF2)*	*612517	PCD 10	Dynein, axonemal, assembly factor 2	14	PCD, KS	NM_018139.2	2514 (837aa)	3.6% ^{Ref 4}
*DNAH5*	*603335	PCD 3	Dynein, axonemal, heavy chain 5	5	PCD, KS	NM_001369.2	13872 (4623aa)	28% ^{Ref 5}
*DNAH11*	*603339	PCD 7	Dynein, axonemal, heavy chain 11	7	PCD, KS	NM_003777.3	13569 (4522aa)	~1% ^{Ref 6}
*DNAI1*	*604366	PCD 1	Dynein, axonemal, intermediate chain 1	9	PCD, KS	NM_012144.2	2100 (699aa)	10% ^{Ref 7}
*DNAI2*	*605483	PCD 9	Dynein, axonemal, intermediate chain 2	17	PCD, KS	NM_023036.4	1818 (605aa)	2-3% ^{Ref 8}
*RSPH4A*	*612647	PCD 11	Radial spoke head protein 4 homolog A	6	PCD	NM_001010892.2	2178 (725aa)	2% ^{Ref 12}
*RSPH9*	*612648	PCD 12	Radial spoke head protein 9 homolog	6	PCD	NM_152732.4	831 (276aa)	~1% ^{Ref 12}
*TXNDC3*	*607421	PCD 6	Thioredoxin domain containing 3	7	PCD, KS	NM_016616.4	1767 (588aa)	4% ^{Ref 13}
*OFD1*	*300170	N/A	Oral-facial-digital syndrome 1	X	PCD, MR	NM_003611.2	3036 (1011aa)	<1% ^{Ref 10}
*RPGR*	*312610	N/A	Retinitis pigmentosa GTPase regulator	X	PCD, RP	NM_000328.2	2448 (815aa)	<1% ^{Ref 11}
*CFTR*	*602421	N/A	Cystic fibrosis transmembrane conductance regulator	7	CF	NM_000492.3	4440 (1480aa)	N/A
Total							50751 (16906aa)	~56-59.6%

Mutation Detection Rate

This Ambry test identifies >92.6% of described PCD mutations (analytic sensitivity). Approximately 56-59.6% of patients with PCD and PCD-Related Disorders have a detectable mutation in the listed sequencing panel genes (clinical sensitivity).

Billing Codes

Test Code	Technique
8122	PCD Panel

Updated CPT Codes for 2013

Turnaround Time

Technique	Days
PCD Panel	42-70

Specialty

Genes

Techniques

Next-Generation (Next-Gen) Sequencing

Specific Mutation Analysis

Panels

Primary Ciliary Dyskinesia Next-Gen Sequencing Panel (includes CFTR)

References

1.     Merveille et al. CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs. Nature Genet. 43: 72-78, 2011. [PMID: 21131972]

2.     Becker-Heck et al. The coiled-coil domain containing protein CCDC40 is essential for motile cilia function and left-right axis formation. Nature Genet. 43: 79-84, 2011. [PMID: 21131974]

3.     Mazor et al. Primary ciliary dyskinesia caused by homozygous mutation in DNAL1, encoding dynein light chain 1. Am. J. Hum. Genet. 88: 599-607, 2011. [PMID: 21496787]

4.     Omran et al. Ktu/PF13 is required for cytoplasmic pre-assembly of axonemal dyneins. Nature. 456: 611–6, 2008. [PMID: 19052621]

5.     Olbrich et al. Mutations in DNAH5 cause primary ciliary dyskinesia and randomization of left-right asymmetry. Nat Genet. 30: 143–4, 2002. [PMID: 11788826]

6.     Schwabe et al. Primary ciliary dyskinesia associated with normal axoneme ultrastructure is caused by DNAH11 mutations. Hum Mutat. 29: 289–98, 2008. [PMID: 18022865]

7.     Zariwala et al. Mutations of DNAI1 in primary ciliary dyskinesia: evidence of founder effect in a common mutation. Am J Respir Crit Care Med. 174: 858–66, 2006. [PMID: 16858015]

8.     Loges et al. DNAI2 mutations cause primary ciliary dyskinesia with defects in the outer dynein arm. Am J Hum Genet. 83: 547–58, 2008. [PMID: 18950741]

9.     Duquesnoy et al. Loss-of-function mutations in the human ortholog of Chlamydomonas reinhardtii ODA7 disrupt dynein arm assembly and cause primary ciliary dyskinesia. Am J Hum Genet. 85: 890–6, 2009. [PMID: 19944405]

10.   Budny et al. A novel X-linked recessive mental retardation syndrome comprising macrocephaly and ciliary dysfunction is allelic to oral-facial-digital type I syndrome. Hum Genet. 120: 171–8, 2006. [PMID: 16783569]

11.   Moore et al. RPGR is mutated in patients with a complex X linked phenotype combining primary ciliary dyskinesia and retinitis pigmentosa. J Med Genet. 43: 326–33, 2006. [PMID: 16055928]

12.   Castleman et al. Mutations in radial spoke head protein genes RSPH9 and RSPH4A cause primary ciliary dyskinesia with central-microtubular-pair abnormalities. Am J Hum Genet. 84: 197–209, 2009. [PMID: 19200523]

13.   Duriez et al. A common variant in combination with a nonsense mutation in a member of the thioredoxin family causes primary ciliary dyskinesia. Proc Natl Acad Sci U S A. 104: 3336–41, 2007. [PMID: 17360648]

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Primary Ciliary Dyskinesia Next-Gen Sequencing Panel (includes CFTR)