The genetics of neonatal respiratory disease

Seminars in Fetal & Neonatal Medicine (2005) 10, 271e282

www.elsevierhealth.com/journals/siny

The genetics of neonatal respiratory disease

Howard Clark a,*, Lucy Side Clark b

a MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford,South Parks Road, and Neonatal Unit, John Radcliffe Hospital, Headington, Oxford OX1 3QU, UKb Oxford Radcliffe Hospitals NHS Trust, Oxford, UK

KEYWORDSRespiratory distresssyndrome (RDS);Bronchopulmonarydysplasia;Surfactant proteins;SP-A;SP-B;SP-C;SP-D;ABCA3;Alveolar proteinosis;Transient tachypnoeaof the newborn (TTN);Cystic fibrosis (CF)screening;Primary ciliarydyskinesia (PCD)

Summary This chapter reviews some of the genetic predispositions that maygovern the presence or severity of neonatal respiratory disorders. Respiratorydisease is common in the neonatal period, and genetic factors have been implicatedin some rare and common respiratory diseases. Among the most common respiratorydiseases are respiratory distress syndrome of the newborn and transient tachypnoeaof the newborn, whereas less common ones are cystic fibrosis, congenital alveolarproteinosis and primary ciliary dyskinesias. A common complication of neonatalrespiratory distress syndrome is bronchopulmonary dysplasia or neonatal chroniclung disease. This review examines the evidence linking known genetic contribu-tions to these diseases. The value and success of neonatal screening for cysticfibrosis is reviewed, and the recently characterised contribution of polymorphismsand mutations in the surfactant protein genes to neonatal respiratory disease isevaluated. The evidence that known variability in the expression of surfactantprotein genes may contribute to the risk of development of neonatal chronic lungdisease or bronchopulmonary dysplasia is examined.� 2005 Elsevier Ltd. All rights reserved.

Genetic factors and the severity ofrespiratory distress syndrome

There are a number of genetic and environmentalfactors that have been clearly identified as affect-ing the severity of neonatal respiratory distress

* Corresponding author.E-mail address: [email protected] (H. Clark).

1744-165X/$ - see front matter � 2005 Elsevier Ltd. All rights resedoi:10.1016/j.siny.2005.02.004

syndrome (RDS). The frequency of RDS is known tobe inversely related to gestational age and birthweight. Caesarean section is also a well-known riskfactor that can favour the development of RDS. Ithas long been observed that male infants fareworse than female infants1 and that Caucasiansfare worse than infants of black or African-Amer-ican ethnicity.2 Advanced maternal age is a riskfactor, although this may be explained by the

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272 H. Clark, L.S. Clark

increased frequency of maternal disease (e.g.hypertension, diabetes, placenta praevia or abrup-tion) in older women, which is associated withmore frequent preterm deliveries and caesareansection.3

Evidence for the importance of genetic factorsin determining the susceptibility to or severity ofRDS has been suggested by twin studies. In a ret-rospective study, the files of twins born between1976 and 1995 at the University of Amsterdam, TheNetherlands were investigated, the twins studiedhaving a gestational age of 30e34 weeks, and oneor both having RDS. RDS occurred more frequentlyin both twins when the twins were monozygotic (12of 18; 67%) than when the twins were dizygotic (18of 62; 29%).4

This supports the notion of a genetic contribu-tion to RDS. However, few formal twin studies havebeen undertaken that aim to define specificgenetic risk factors. Linkage studies have not sofar helped in identifying genetic susceptibilities toRDS because large family histories with multipleaffected individuals are not available, given thatmany affected infants have died in infancy. Thecandidate gene approach is one method by whichsuch associations can be established.

Candidate gene susceptibility factorsfor respiratory distress syndrome andbronchopulmonary dysplasia

Lung surfactant is a complex of phospholipidsand proteins responsible for maintaining alveolarstability, and lung surfactant deficiency causesRDS. Candidate genes that could potentiallyaffect RDS risk therefore include genes of thesurfactant system, genes closely involved in theprocesses of postnatal adaptation and the manygenes that may be involved in regulation ofpulmonary inflammatory responses and develop-ment.

Candidate genes thus include those encodingthe four surfactant apoproteins, SP-A, SP-B SP-Cand SP-D, as well as the genes that in part affectthe expression and regulation of important sur-factant components (e.g. thyroid transcriptionfactor-1). This review focuses on the genes forSP-A, SP-B, SP-C and SP-D themselves. The risk ofbronchopulmonary dysplasia increases with theseverity of RDS, so candidate genes affecting RDSseverity are also likely to confer an increased riskof developing bronchopulmonary dysplasia or neo-natal chronic lung disease (CLD).

SP-A

SP-A, like SP-D, belongs to a family of proteinsnamed collectins because they have collagenousand lectin-binding domains. SP-A is the mostabundant surfactant associated protein and wasthe first to be described. Central roles in thestructure (tubular myelin formation),5 metabolismand function7,8 of surfactant, as well as in hostdefence,9 have been attributed to SP-A. It hasbecome accepted that low SP-A levels in respira-tory secretions from premature infants are associ-ated with more severe RDS and the subsequentdevelopment of CLD.10

Like the structurally similar and highly homolo-gous serum collectin, mannan-binding lectin(MBL), SP-A is a large protein composed of up to18 polypeptide chains, each of about 30 kDa. MBLis a molecule of innate immunity that binds tomicroorganisms, enhances their phagocytosis andcan activate the complement system. SP-A alsobinds microbes and promotes phagocytosis butlacks the ability to activate complement, interact-ing with immune cells via receptors.

Human SP-A is composed of two similar butdistinct types of polypeptide chain, designated a2and a3.11 Subunits of SP-A are each built up by theassociation of three polypeptide chains, thought tobe two SP-A gene I translation products (SP-A a3chain) and one SP-A gene II product (SP-A a2chain).12 The collagenous regions of these poly-peptides intertwine to form a collagen triple helix.The non-collagenous regions form a globular‘head’ consisting of three lectin-binding domains.A total of six such subunits may associate to formthe characteristic ‘bunch of tulips’ structure. Inthis structure, the globular heads form the ‘flow-ers’ and the collagen helices form the ‘stalks’.13

Isolated SP-A has been regarded as being mainlyhexameric in structure, made up of six subunitseach with three polypeptides, as with the highlyhomologous structure of MBL.

This oligomerisation greatly increases the over-all avidity of the otherwise weak lectin-mediatedinteractions of individual SP-A subunits with car-bohydrate targets on pathogens. The trimericsubunits of the collectins have only limited affinity(mM) for carbohydrate targets, but their oligo-meric assembly provides a high avidity so that theoligomeric proteins bind to ligands selectively withhigh affinity (nM).

Thus, the structure of SP-A means that, like thecase of MBL, it is potentially vulnerable to pointmutations in the collagen domain that exert a dom-inant negative effect on protein function due to

Genetics of neonatal respiratory disease 273

a loss of the ability of the protein to oligomerise.While these mutations in MBL have been associatedwith an increased risk of infection in infancy andchildhood,14 to date no such mutations in SP-A havebeen described in humans.

SP-A is not the primary protein determiningsurfactant function but acts to enhance surfactantactivity. For example, SP-A protects surfactantactivity against the inhibitory effects of serum.15,16

SP-A inhibits surfactant secretion by type II cells inculture and facilitates the type II cell uptake ofphospholipid liposomes.6

Highly specific interactions between type II cellsand SP-A are known to be involved in the phospho-lipid uptake processes.17 SP-A-lipid interactionsinvolving a lipid-binding site around the Arg197residue of SP-A have a profound effect on surfac-tant lipid recycling. Single point mutations thatchanged the Arg197 of SP-A to either Lys or Hisdramatically altered lipid uptake by type II cells.The Lys197 mutation exhibited a two-fold increasein lipid uptake activity, whereas the His197 muta-tion displayed all SP-A functions studied except forlipid uptake. No such naturally occurring mutationsaffecting surfactant turnover have been described.

Indeed, how important the properties of SP-Aaffecting surfactant structure and function are invivo has been questioned because SP-A knockoutmice have apparently normal surfactant function18

and only minor changes in surfactant metabo-lism.19 It is therefore likely that there is redun-dancy in the system that allows for the criticalfunctions of surfactant to be maintained even inthe absence of SP-A.

Although SP-A has negligible surface activitywhen mixed with phospholipids in the absence ofother proteins, it markedly accelerates phospho-lipid film formation in the presence of SP-B.8,20

This synergistic activity is associated with the SP-A-dependent formation of the highly orderedsurfactant structure known as tubular myelin.The marked cooperative effect of SP-A on SP-Bactivity is most evident at low phospholipid con-centrations7,8 (such as would be the case in thesurfactant-deficient premature lung).

Genetic studies linking SP-A andrespiratory distress syndrome orchronic lung disease

There are two genes for human SP-A, which areclosely linked and highly polymorphic, residing on10q22eq23. Several studies have found associa-tions between SP-A polymorphisms and severity ofRDS. In a study by Martilla et al, the main SP-A1allele 6A2 (PZ0.030), genotype 6A2/6A2

(PZ0.0042) and haplotype 6A2-1A(0) (PZ0.016)were overrepresented in healthy premature twininfants compared with twins with RDS.21 Thehomozygous genotype 6A2/6A2 was overrepre-sented in twin pairs of whom both were healthycompared with twins concordant for RDS (OR 0.18,CI 0.06e0.60, PZ0.0016) and born between 32 and36 weeks’ gestation. 6A2/6A2 was also overrepre-sented in healthy twin pairs with a birth weightsum higher than the median (OR 0.15, CI 0.04e0.60, PZ0.0025).21

In twins, the association between SP-A poly-morphism and RDS is different from that seen inpremature singleton infants. The factor associatedwith SP-A genotype-specific susceptibility to RDSappears to be related to the size of uterus and thelength of gestation at birth. This latter finding isinteresting, in view of the recent observation thatSP-A may also be an important factor involved intriggering parturition.22

The cooperative properties of SP-A and SP-B insurfactant function and structure are consistentwith the results of other genetic studies exam-ining the combined effect of SP-A and SP-Bpolymorphisms on risk for RDS. Three studiescarried out by Marttila et al. report that certainpolymorphisms of SP-A and SP-B in prematuretwins21,23 confer an increased risk of RDS oraffect RDS severity. There was an associationbetween the SP-B Ile131Thr polymorphism andRDS. The threonine allele was associated withthe risk of RDS, particularly in the first-born twininfants.23

In one study by Marttila et al., 441 prematuresingleton infants and 480 twin or multiple infantswere genotyped for SP-A1, SP-A2 and SP-B exon 4polymorphisms and intron 4 size variants in a ho-mogeneous white population. The SP-A 6A2 allelein the SP-B Thr131 background predisposed thesmallest singleton infants to RDS, whereas near-term multiples were protected from RDS. Therewas thus a combined effect of SP-A and SP-Balleles on the risk of RDS.24

Although no mutations of the translated portionsof the SP-A genes have been reported in humans,these genetic studies show that certain SP-A poly-morphisms are clearly associated with an increasedseverity of RDS. At least one study has linked theseas susceptibility factors in the development ofneonatal chronic lung disease (CLD).25

The role of SP-A in host defence is stronglysupported by studies of SP-A knockout mice, whichhave been shown to be susceptible to infectionwith a large number of pathogens, including groupB streptococci, Pseudomonas, Staphylococcusand respiratory syncytial virus (RSV).26e28 It is


therefore becoming clear that SP-A may have dualroles in the alveolar lining e both in promotingsurfactant function and in innate immunity e andit is possible, but unproven, that these functionsare differentially regulated through a differentialexpression of the two SP-A genes.29 Since infectionand inflammation are well-recognised factors af-fecting the severity of RDS and the development ofCLD, the host defence properties of SP-A mayprovide another mechanism to explain why certainSP-A polymorphisms are associated with moresevere RDS or with CLD.

Hydrophobic SP-B and SP-C

In contrast to the hydrophilic SP-A and SP-D, thehydrophobic surfactant proteins, SP-B and SP-Cdirectly affect the biophysical properties of sur-factant lipids both in vivo and in vitro, and arecritically important for surfactant function. Rapidadsorption of surfactant phospholipid to the air-liquid interface is known to be critical for main-taining the stability and morphological integrity ofthe alveolus.30,31 SP-B, SP-C or mixtures of the twoproteins markedly enhance the rate of formationof a phospholipid surface film at an air-liquidinterface in vitro. In vivo, preparations of SP-B,SP-C and surfactant lipids have been shown toincrease lung compliance and preserve the in-tegrity of the distal airways in prematurely de-livered, ventilated fetal rabbits.30e32 Infants withRDS treated with surfactant preparations contain-ing SP-B and SP-C show a significant improvementin oxygenation and have lower ventilatory require-ments and decreased mortality rates.33 Low levelsof SP-B and SP-C have been observed in trachealfluid from infants with RDS.

As indicated above, some studies have exploreda variable tandem repeat region in intron 4 of SP-Band found higher rates of variant alleles in pop-ulations of infants with RDS compared with controlinfants.34e36 As observed by Nogee,37 such associ-ations have generally been with all variant allelesgrouped and compared with the most commonallele, rather than on the risk associated witha specific allele, and have not always controlledfor genetic background. There is no known associ-ation of these intronic variants with variation in SP-B gene expression, processing or function, and thusthe precise mechanism by which they might in-fluence the severity of RDS is not clearly under-stood. The threonine allele of the SP-B Ile131Thrpolymorphism (discussed above) was associatedwith RDS23 and has also been found to be associatedwith an increased risk for ARDS in adult subjects.38

Although this alters a potential glycosylation site inpro-SP-B, and in vitro experiments have confirmedthat this polymorphism does affect the glycosyla-tion of pro-SP-B, there is no evidence that it isimportant in SP-B folding or processing to maturefunctional SP-B, and thus it is currently unclearprecisely how such a change affects RDS severityand pathogenesis.

By contrast with SP-A, definitive genetic evi-dence of the critical importance of both thehydrophobic proteins SP-B and SP-C in maintaininglung function at the alveolar air-water interface isamply demonstrated by individuals with single-gene mutations causing a deficiency of SP-B orSP-C. A deficiency of either of these proteins leadsto severe lung disease.

Hereditary deficiency of human SP-B

Mutations in the gene for SP-B resulting in SP-Bdeficiency cause a lethal neonatal respiratorydisease, hereditary SP-B deficiency.39 The genefor SP-B resides on human chromosome 2. ThemRNA is initially translated into a pre-pro-proteinof 381 amino acid residues, which yields a pro-protein after co-translational excision of the first23 amino acid signal peptide. Further processingremoves amino acids at both the C- and N-terminalends to yield the highly hydrophobic 79 amino acidmature SP-B, a disulphide linked homodimer of the79-residue polypeptide chains, each 8.7 kDa.40

SP-B is essential to surfactant structure (specif-ically for tubular myelin formation) and is essentialin the intracellular processing of surfactant. TypeII cells of SP-B knockout mice contain numerousmultivesicular bodies but no mature lamellarbodies, suggesting that the formation of mem-brane lamellae is SP-B dependent.41 SP-B is alsoessential for the correct processing and secretionof the mature SP-C protein, so that SP-B knockoutmice or infants with congenital SP-B deficiencyalso lack mature SP-C. The clinical phenotype isusually a term infant with a disease resemblingsevere RDS. The course can initially be mild,depending on residual SP-B expression or theresidual function of expressed pro-SP-B, whichmay vary from 5% to 10% depending on the precisegenetic defect.

Hereditary SP-B deficiency has resulted frommutations in the coding regions of the SP-B geneor mutations likely to cause aberrant splicing. It isalso possible, although not yet proven, that (giventhe complex processing needed to generate ma-ture SP-B) mutations in genes essential to pro-cessing could result in the SP-B deficiencysyndrome, since cases have been identified in


which no mature SP-B is detectable although nomutation in the SP-B gene is present.42 The mostcommon mutation, a 121ins2 mutation, inducesa frameshift leading to undetectable mature SP-Blevels in homozygotes and variable SP-B expres-sion in heterozygotes. Numerous other mutationshave been discovered, but the 121ins2 mutationaccounts for up to 66% of cases. SP-B deficiencyis, however, a rare syndrome, the most commonmutation having an estimated gene frequency of1 in 1000 individuals and the estimated diseaseincidence being 1 in 1.5 million births.42 Althoughinitially responsive to surfactant therapy, trueSP-B deficiency generates respiratory distress thatis invariably fatal unless treated by lung trans-plantation.

SP-C and respiratory distress syndrome

SP-C, like SP-B, is a small hydrophobic protein.SP-C is unique to pulmonary surfactant and has noknown homologues. SP-C contains covalentlylinked fatty acyl chains and is one of the mosthydrophobic natural polypeptides known.43 Thehuman SP-C gene has been localised to the shortarm of chromosome 8.44 The SP-C mRNA ofapproximately 0.9 kb encodes a pre-protein of197 amino acids.44 The SP-C pro-protein does notcontain an N-terminal signal peptide. It is pre-sumed that the 23-residue N-terminal and 133e139residue C-terminal peptides of the SP-C pro-pro-tein maintain the highly hydrophobic active pep-tide in solution within the cell.

Processing of the SP-C pro-protein is linked toSP-B expression as indicated by the phenotype ofSP-B-deficient human infants and mice.39,41,45 Inthe absence of SP-B expression, the SP-C pro-protein is incompletely processed, resulting in anaccumulation of SP-C peptide with a molecularweight of approximately 12 kDa.46 A possiblerelationship between SP-C expression and RDSwas suggested by the finding of absent or markedlyreduced SP-C expression in a strain of Belgiancalves that died of RDS.47

Polymorphisms of the SP-C gene have beenexamined for any association of the allelic variantswith susceptibility to RDS and bronchopulmonarydysplasia in a Finnish population.48 Three biallelicpolymorphisms of the SP-C gene’s exons 1, 4 and 5were identified that encode pro-SP-C. The fre-quencies of these polymorphisms were evaluatedin a study population consisting of 158 DNA samplesfrom full-term infants. In addition, the linkagedisequilibrium between the SP-C alleles was eval-uated by a haplotype analysis of parent-infant

triplets. SP-C polymorphisms were associated withRDS and with very premature birth. The strengthof allelic associations differed according to thegender of the premature infants.

In addition to these studies of SP-C polymor-phism, further genetic evidence for the impor-tance of SP-C has been derived from the discoverythat mutation of the SP-C genes leads to intersti-tial lung disease (ILD).

Lung SP-C deficiency

Unlike SP-B gene mutations, which lead to re-spiratory distress very soon after birth, SP-C de-ficiency usually presents at a few months of age.The SP-C gene, located on human chromosome 8,is highly conserved.49 Insights into various roles ofSP-C have recently been provided by studies inpatients with a selective deficiency of the SP-Cgene or dominantly inherited mutations in the SP-Cgene. An SP-C mutation involving a single intronicbase substitution led to a skipping of exon 4 anda shortening of the SP-C pro-protein produced. Adominantly inherited mutation in the SP-C gene isassociated with ILD.50 ILD includes a heterologouscollection of uncommon disorders detected inindividuals who present with progressive lungdisorders associated with frequent pulmonary in-fections, exercise limitation, tachypnoea andshortness of breath. In general, ILD is associatedwith alveolar inflammation, pulmonary infiltrationwith monocytes and macrophages, a progressiveloss of alveolar structure and pulmonary fibrosis.As well as deficiency in SP-C, mutations in thecoding regions of the SP-C gene may lead tointracellular trafficking defects of the SP-C proteinand, depending on the location of the mutation inthe SP-C protein-coding gene sequence, result indifferent patterns of transport defects and lungpathology.51

These mutations were first identified in a familyin which there was a history of several siblingsbecoming oxygen dependent at age 3e4 monthsand developing ILD and pulmonary fibrosis. Thereis generally no history or detectable evidence ofviral infection, and SP-C is undetectable in thelung tissue on biopsy.

Lung SP-D

The possibility of profound genetic influences onrespiratory disease in the human neonate re-lating to the expression of hydrophilic surfactantproteins genes (SP-A and SP-D) has been


highlighted by studies of the phenotypes ofsurfactant protein gene knockout mice. In par-ticular, the findings that SP-A-deficient mice aremore susceptible to infection and also that SP-Dknockout mice develop a chronic low-grade lunginflammation and ultimately develop pulmonaryemphysema indicate an important role in vivo forthese proteins. The possible relevance of theseas a contributory pathway for the developmentof neonatal CLD has recently been reviewed.52

SP-D is structurally homologous to SP-A andMBL. Three of the 43 kDa polypeptide chainsintertwine to form monomeric subunits thatassociate into oligomers. In vitro experimentssuggest that SP-D, like SP-A, is involved in thefirst line of defence against inhaled pathogens(reviewed in ref.9) This is consistent with thefact that 90e95% of SP-D readily dissociates fromlipids and is present in solution, available to actas a soluble opsonin in the alveolar lining layer.

Unlike the case with SP-A, there are no reportsof SP-D polymorphisms conferring a higher risk ofRDS or of neonatal bronchopulmonary dysplasia.However, low SP-D levels in tracheal secretions inthe first few days of life from preterm infantswere linked with an increased risk of neonatalCLD.53 Only a small number of SP-D polymor-phisms have been characterised. Certain SP-D(and SP-A) alleles have been linked to possiblesusceptibilities to chronic obstructive pulmonarydisease in a Mexican population,54 and both SP-Aand SP-D polymorphisms are associated with in-creased severity of childhood infection withRSV.55e57 Three biallelic SP-D gene polymorphismswere genotyped in DNA samples from 84 infantswith bronchiolitis and 93 healthy age-, gestationalage- and sex-matched controls. Significant differ-ences were observed in the SP-D allele frequen-cies for amino acid 11 between the RSV infantsand their matched controls. The frequency of theallele coding for Met 11 (PZ0.033) was increasedin the severe RSV group. The frequency of thehomozygous genotype Met/Met for amino acid 11was increased in the RSV group relative to thecontrols, whereas the heterozygous genotypetended to be less frequent among the RSV casesthan in the matched controls.55

SP-A and SP-D polymorphisms andinflammatory lung disease

Although there is now plentiful evidence from bothin vitro and in vivo studies supporting the role ofSP-A and SP-D in defence against infection and inmodulating inflammation and allergic processes,

their potential importance and relevance tohuman ILD has only recently come under investi-gation. It is emerging that polymorphisms in theMBL, SP-A and SP-D genes are indeed associatedwith susceptibility to respiratory disease inhumans in infants and also adults.

The best characterised are polymorphisms inthe MBL gene. MBL has three replacement single-nucleotide polymorphisms in exon 1 of the MBLgene: D52C, G54D and G57E.14 These single-nucle-otide polymorphisms disrupt the collagen helix andhence the assembly of trimers, and therefore actas dominant mutations resulting in profound re-ductions in high-order MBL oligomers. The pres-ence of these low-producing coding alleles istermed ‘O’. O/A individuals might have one-eighththe level of some A/A individuals.

Several studies have now convincingly demon-strated an association between low-coding MBLalleles and increased risk of infection. The hypoth-esis would predict that low or undetectable levelsof MBL predispose the host to infections. The initialidea was that an opsonic defect in serum thatcorrelated with a broad phenotype of recurrentinfectionwas due to a lack of MBL in young children.Later, Turner modified this concept and suggestedthat the phenotype of susceptibility to infectionwas more obvious if there was an associated defectin adaptive or innate immunity.14 One examplewould be that an antibody isotype deficiency inconjunction with low MBL levels would collectivelypresent the host with an increased risk to infection.

Garred and colleagues demonstrated that pa-tients with cystic fibrosis who also inherit low-secretor MBL haplotypes have a reduction in lifeexpectancy of 5-8 years.58 Although the exactmechanism is not clear, one plausible explanationis that these patients’ lungs are colonised earlierwith Burkholderia cepaciae and Pseudomonas aer-uginosa. Once this occurs, a refractory cycle oflung injury begins that ultimately results in thedemise of the patient. It would be of great interestto investigate the possibility of SP-A or SP-D poly-morphisms contributing to the severity of cysticfibrosis. The investigation of further links betweenSP-A and SP-D polymorphisms in unexplainedchronic ILD in infancy and childhood is an arearipe for further investigation.

Pulmonary alveolar proteinosis

Congenital alveolar proteinosis syndromes arecharacterised by the accumulation of surfactantmaterial in the alveolar space. SP-B deficiency, asdescribed above, leads to alveolar proteinosis, but


there are likely many other causes e both congen-ital and acquired e which may lead to similarpulmonary pathology. For example, a deficiency ofthe common beta chain of receptors for granulo-cyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 and interleukin-5 has beenidentified as a potential mechanism for pulmonaryalveolar proteinosis in four young infants.59 Inadult populations, pulmonary alveolar proteinosisis frequently caused by autoantibody formationagainst GM-CSF.60 Other genetic causes of thissyndrome will probably be characterised in thefuture, but in the majority of cases, the causecurrently remains unknown.

ABCA3 deficiency and lung disease

ABCA3 is a member of the ATP-binding cassettefamily, which are transmembrane proteins involvedin membrane trafficking, The ABCA subgroup ofproteins is predominantly involved in lipid transportacrossmembranes. The gene for ABCA3 is expressedin type II cells, and the protein localises to lamellarbodies, suggesting that it may have an importantrole in surfactantmetabolism. It is closely related toABCA1 and ABCA 4e proteins that are known to playan important part in phospholipid turnover andmetabolism and for which disease-causing muta-tions have been identified.

Mutations in ABCA1 cause Tangier disease, a dis-order causing cholesterol accumulation in macro-phages and peripheral tissues with an absence ofhigh-density lipoproteins. A candidate gene ap-proach was employed to assess whether mutationsin ABCA3 were able to explain surfactant deficiencyin full-term infants with respiratory distress ofunknown origin.61 Mutation of the ABCA3 genecauses fatal surfactant deficiency in newborns.Histopathological changes were consistent withdesquamative interstitial pneumonitis and resem-bled neonatal alveolar proteinosis. There wasa hyperplasia of type II cells with abnormal lamellarbodies, an increased accumulation of alveolarmacrophages in the distal airspaces and varyingamounts of proteinaceous material in the alveolarspace, with interstitial thickening. Although theincidence of these mutations in the general pop-ulation is unknown, it is possible that this is a morecommon cause of respiratory failure in the new-born than surfactant protein mutations.

Transient tachypnoea of the newborn

Transient tachypnoea of the newborn (TTN) isa common cause of respiratory distress in the

neonatal period, characterised by an oxygen re-quirement and tachypnoea, and occurring in 1e2%of term babies. The clinical course is usuallybenign, recovery occurring spontaneously withina few days. Recognised risk factors for TTN includeprematurity, caesarean section, those whosemothers were affected by maternal diseases, malegender and being a twin.3

It is considered to be due in part to defectivemechanisms of lung liquid clearance after birth, andsome others consider that mild and transient sur-factant deficiency may contribute to the disease.Candidate genes that may confer a susceptibility toTTN in addition to the SP genes include other genesaffecting lung liquid clearance. Examples are genesaffecting the activation or increased expression ofsodium transport molecules, such as the epithelialsodium channel or the Na(C)-K(C)-ATPase pump,possibly the cystic fibrosis transmembrane conduc-tance regulator (CFTR).62 Animal studies have sug-gested a role for keratinocyte growth factor63 ortransforming growth factor alpha,64 and a minorrole for genes regulating aquaporins.65

However, only one genetic study so far hasexamined a population of infants with TTN lookingfor associations with SP polymorphisms or muta-tions. Tutdibi and co-workers sought to establishwhether there was an association between intron 4polymorphisms of SP-B or heterozygosity for theSP-B 121ins2 mutation and TTN in a population ofterm babies presenting with TTN in universityhospitals in Germany and Turkey.66 DNA wasobtained from 83 health term controls and 75infants suffering from TTN. None of the patientswere heterozygous for the SP-B 121ins2 mutation,and the frequency of intron 4 variations did notdiffer between healthy babies and newborns withTTN. Genetic studies for associations with othercandidate genes affecting TTN have not so farbeen carried out.

Cystic fibrosis

Cystic fibrosis is a common autosomal recessivedisorder. It is caused by mutations in the CFTRgene, located at chromosome 7q31.2. CFTR con-tains 27 coding exons transcribing a 6.5 kb mRNA.This gene functions as a chloride channel in thecell membrane. Biallelic mutations in CFTR lead tothickened exocrine secretions, giving rise to theclinical manifestations of cystic fibrosis. Heterozy-gous carriers are unaffected.67 There is consider-able ethnic variation in the prevalence of cysticfibrosis (Table 1).


The clinical features of classic cystic fibrosis arewell described. It is a multisystem disorder, butthe major cause of morbidity and mortality ispulmonary disease. Treatment of the infectionand chronic inflammation that leads to end-stagelung disease remain a challenge for physicians.However, life expectancy for affected individualsis increasing with improved clinical care. Over 85%of cystic fibrosis sufferers will have exocrinepancreatic insufficiency. Some (15% or more) willgo on to develop cystic fibrosis-related diabetesmellitus, generally from adolescence onwards.Liver disease may vary from elevated liver en-zymes (4e5%) in childhood to biliary cirrhosis ina small number of individuals in adulthood. Themajority of males (more than 95%) are infertile,although females are fertile. Mutations in CFTR arelinked with a number of other conditions, includingcongenital bilateral absence of the vas deferens,pancreatitis, liver disease and disseminated bron-chiectasis.67

There are currently over 1300 causative muta-tions in CFTR listed on the Cystic Fibrosis MutationDatabase.68 However, the 33 most common muta-tions account for over 90% of all mutations de-tected in clinical testing. Thus, medical geneticslaboratories will generally offer a test that willpick up most, but not all, CFTR mutations. As thefrequency of individual mutations varies depend-ing upon ethnic origin, the detection rate forgenetic testing in individuals may also vary, de-pending upon the test used. In Caucasian popula-tions, DF508 accounts for 75% of all mutations.Conversely, in individuals of Ashkenazi Jewishorigin, W1282X accounts for approximately 60% ofthe total.67

Genotype-phenotype correlations in cystic fi-brosis have been well documented.67,69e71 TheR117H mutation is generally associated with pan-creatic sufficiency and a milder phenotype. Varia-tions in the size of the polythymidine (polyT) tractin intron 8 can also affect phenotype. The 5T alleleis a very mild CFTR mutation, whereas the 7T and9T alleles are normal. Men with congenital bi-lateral absence of the vas deferens (without other

Table 1 Variation in the prevalence of cysticfibrosis

Ethnic variation Approximatedisease prevalence

Carrierincidence

White Caucasian 1/2500 1/25Afro-Caribbean 1/15,000 1/61Hispanic 1/8500 1/46Ashkenazi Jew 1/3500 1/29Anglo-Asian 1/10,000 1/50

features of cystic fibrosis) who are homozygous forthe 5T allele have been reported. In addition, the5T allele may modify the phenotype of the R117Hmutation. Although the majority of CFTR muta-tions occur in cis (i.e. on the same chromosome)with the 7T and 9T alleles, R117H may occur in ciswith 5T or 7T. As expected, R117H-7T is generallymilder than R117H-5T.

Cystic fibrosis screening in theneonatal period

Cystic fibrosis is difficult to diagnose in the neo-natal period. It may be suspected antenatally onan anomaly scan due to the presence of echogenicbowel. These families are generally offered testingfor cystic fibrosis, although probably less than 5%of all echogenic bowel represents cystic fibrosis. Itis most commonly detected owing to the gutmanifestation of meconium ileus rather than pre-senting with respiratory symptoms. Although only10e15% of individuals with cystic fibrosis willpresent with meconium ileus, it is virtually patho-gnomonic of the condition. Infants with cysticfibrosis may also present with hyponatraemiaand/or hypoproteinaemia.67

The difficulty of diagnosing cystic fibrosis in theneonatal period has led to suggestions that neo-natal screening should be carried out for thisdisease. A prerequisite for screening should be thatearly intervention is effective or at least that theinformation from screening can influence decisions.There has been considerable debate over whetherthe widespread introduction of neonatal screeningfor cystic fibrosis will have benefit,72,73 but it hasbeen supported by patient organisations such as theCF Trust. Screening for cystic fibrosis is currentlybeing piloted in a number of centres and is likely tobe extended throughout the U.K.

Neonatal screening will comprise testing ofimmunoreactive trypsin (IRT) levels on Guthriespots. IRT levels at the top 0.5e0.6% of thepopulation will necessitate further investigation,although the majority of these infants will nothave cystic fibrosis. Most laboratories will thenperform CFTR mutational analysis on the bloodspot, by testing for either DF508 or a wider panelof CFTRmutations. If two mutations are found, theinfant will be referred to the local cystic fibrosisservice. If one mutation is found, repeat IRTtesting will lead to a sweat test or similar di-agnostic test if it remains abnormal.

There are potential benefits of neonatal screen-ing in terms of reducingmorbidity from lung diseaseand pancreatic insufficiency. Families may receivegenetic counselling earlier, allowing them to make


decisions about genetic testing in future pregnan-cies. This process will inevitably detect cysticfibrosis carriers in childhood, and this raises issuesof informed consent for the test. It has generallybeen thought undesirable to test carrier status inchildhood, but a Welsh study has shown that it wasnot perceived as problematic, at least by parents.74

There are issues about the sensitivity of the test,whether some individuals may be missed on the IRTtest and whether a second mutation will not bepicked up on genetic screening. As already dis-cussed, ethnic variation may affect this, particu-larly if DF508 is used alone. Conversely, milderCFTRmutations may be picked up, depending uponthe screening panel used. These mutations may notnecessarily be associated with childhood disease,and this could potentially lead to discrimination,for example in terms of insurability. In some cases,genetic testing will inadvertently reveal cases ofnon-paternity. It is therefore important that ap-propriate genetic counselling is in place to supportthe screening programme.

Primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD), formerly knownas Kartagener’s or immotile cilia syndrome, isinherited in an autosomal recessive manner inthe majority of cases. Prevalence is estimated at1 in 15,000e20,000, but because PCD is often mild,it is likely that many of these individuals remainundiagnosed. Half of all patients have associatedsitus inversus; dextrocardia alone is not an associ-ation. The main problems in PCD are mucousretention in the lung leading to recurrent infec-tion, and chronic sinus and middle ear infections.There may be decreased fertility in males due toreduced sperm motility or abnormal cilial functionin the vas deferens. In the fallopian tubes, dyski-netic cilia may result in ectopic pregnancy.75,76

PCD can usually be managed successfully inthe outpatient setting with regular chest physio-therapy and prompt treatment of infection withantibiotics. Appropriate therapy should help toprevent deteriorating lung function, and life ex-pectancy approaches normal. Nevertheless, 98% ofall adults and 60% of children will have some degreeof bronchiectasis. The insertion of myringotomytubes tends to result in chronic discharge withoutany improvement in hearing. If speech or learning islikely to be affected by hearing loss, hearing aidsare recommended. The outcome for hearing in thelong term is good. Counselling should be offered topatients with reduced fertility; men may be helpedby intracytoplasmic sperm injection.75,76

Situs inversus may be detected antenatally onan anomaly scan and is estimated to be associatedwith PCD in approximately half of all cases. Pro-spective parents should be counselled accordinglyif this is found. Generally, even if there is dextro-cardia, the heart is structurally normal. However,one study showed more complex congenital cardi-ac defects in a small number of individuals, so itmay be prudent to offer a more detailed fetalcardiac scan. PCD may also present in the neonatalsetting. Persistent cough and/or nasal congestionmay be apparent shortly after birth. Affectedinfants may present with unexplained tachypnoeaor RDS at term. Indeed, neonatal respiratorysymptoms may be present in around three-quar-ters of affected individuals.77

Definitive testing for PCD requires nasal cilialbrush biopsy. The ciliary beat frequency can thenbe assessed; normal frequency is 12.8 Hz inchildren. However, beat frequency may be normalin some patients, necessitating an analysis of thecilial beat pattern as well as frequency, and this isa specialist investigation. Transient damage to thecilia may be caused by colds, and abnormal bi-opsies may need to be repeated. Other tests, suchas nasal nitric oxide measurement, are not cur-rently in widespread use.75,76

Cilia are complex structures present in therespiratory tract (including the sinuses and middleear), ependyma of the brain, vas deferens andfallopian tubes. Briefly, they comprise two centralmicrotubules surrounded by nine pairs of periph-eral microtubules. Inner and outer dynein armsconnect these to form a ‘hub and spoke’ arrange-ment. The dyneins act as the ‘motor’ enabling thecilia to beat. An absence of the dynein arms is oneof several cilial abnormalities seen in PCD.75 Ciliaare also present in the embryonic node and playa crucial role in left-right patterning. The node isa structure that develops at the tip of the embryoduring gastrulation, at the junction of embryonicectoderm and endoderm. The determination oflaterality is regulated by a number of genes thatact at the node; this is reviewed in detail else-where.78 Genetic mutations leading to an absenceof nodal cilia result in bilateral symmetry beingpreserved (e.g. as in Ivemark syndrome), whereasif the mutation affects cilial motility, it results inrandom asymmetry. Thus, mutations resulting inPCD should give rise to situs inversus in 50% ofcases, which seems to be borne out by clinicalobservation.

As the cilia are assembled from many differentproteins, a large number of candidate genes andloci are involved in PCD. To date, only mutations indyneins have been implicated in PCD. The proteins


DNAH5, DNAI1and DNAH11 form part of the outerdynein arm, and mutations in the genes encodingthese have been found in PCD.79e81 There remaina large number of mapped PCD loci at which thecausative genes have yet to be identified, includ-ing a locus at 19q13.4 in five families.82 Interest-ingly, one family with X-linked retinitis pigmentosaresulting from a mutation in the RPGR (retinitispigmentosa GTPase regulator) gene also had a clin-ical phenotype consistent with PCD.83

Because of the genetic heterogeneity in PCD,genetic testing is not offered in these patients inthe clinical setting. Cilial brush biopsy remains thecurrent mainstay of diagnosis. However, findingmutations in families will likely further our un-derstanding of cilial structure and function.

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Practice points and research agenda

� the candidate gene approach has yieldedconsiderable information about specificgenetic risk factors in a range of respira-tory diseases affecting the neonate

� in particular, the investigation of linksbetween respiratory disease and the com-ponents of the human surfactant systemhas led to an increased understanding ofthe pathogenesis of some common andrare respiratory diseases

� it is likely that this will continue to presenta fruitful area of investigation

� understanding the genetic contributions torisk of disease may in future allow betterprognostication and early intervention, aswell as offering the potential of geneticcounselling for recurrence risks in familiesexperiencing severe neonatal respiratorydisease

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The genetics of neonatal respiratory disease