Article Text

Download PDFPDF

Genetic testing in children with surfactant dysfunction
  1. Simona Turcu1,2,
  2. Emma Ashton3,
  3. Lucy Jenkins3,
  4. Atul Gupta4,
  5. Quen Mok1
  1. 1Department of Pediatric Intensive Care, Great Ormond Street Hospital, London, UK
  2. 2Department of General Pediatrics, Evelina Children Hospital, London, UK
  3. 3North East Thames Regional Genetics Laboratory, Great Ormond Street Hospital, London, UK
  4. 4Department of Respiratory Pediatrics, Kings College Hospital, London, UK
  1. Correspondence to Dr Quen Mok, Department of Pediatric Intensive Care, Great Ormond Street Hospital, Great Ormond Street, London WC1N 3JH, UK; quen.mok{at}gosh.nhs.uk

Abstract

Objectives To present the UK experience in genetic diagnoses of surfactant protein dysfunction disorders and develop a referral algorithm for neonates and children with persistent respiratory problems.

Materials and methods Between 2006 and 2011, 427 cases were referred for surfactant mutation analyses to the North East Thames Regional Molecular Genetics Laboratory at Great Ormond Street Hospital, London. The results were reviewed and referring physicians of mutation positive cases contacted to complete a questionnaire providing clinical, radiological, histological and outcome information.

Results 25 new cases were found to have genetic mutations for surfactant dysfunction disorders (7.5%), with six resulting in surfactant protein B dysfunction, seven surfactant protein C dysfunction and 12 ATP-binding cassette subfamily A member 3 (ABCA3) dysfunction. The referrals were from 15 different paediatric centres. In addition, three affected surfactant protein B (SFTPB) cases were prenatal diagnoses, following the birth of previously affected children. The majority of the confirmed cases (23 of 25) were born after 37 weeks gestation. All children with SFTPB dysfunction and the majority of ABCA3 patients presented with respiratory distress at birth. All SFTPB cases died from intractable respiratory failure. The outcome for ABCA3 mutations was variable with seven survivors. The clinical and radiological presentation of surfactant protein C (SFTPC) patients suggested mainly interstitial lung process with the majority surviving on medication.

Conclusions Surfactant mutation analysis is now well established in the UK and allows better genetic diagnosis and counselling. The rarity of the condition makes it difficult to develop a validated algorithm for genetic evaluation with a need for international networking. Referrals need to be rationalised for the service to be time and cost effective.

  • genetic testing
  • surfactant dysfunction
  • paediatric lung disease

Statistics from Altmetric.com

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.

What is already known on this topic

  • Respiratory distress syndrome in term neonates or interstitial lung disease in older children especially with a positive family history can be caused by surfactant dysfunction.

  • Identification of specific mutations in these surfactant genes is now available from peripheral blood.

  • Various genotypes cause overlapping phenotypes and there are limitations of genetic testing with regard to the possible identification of novel variants with unknown significance.

What this study adds

  • Genetic testing for surfactant dysfunction disorders is now available in the UK and this study brings together positive paediatric cases tested in the UK.

  • Rarity of the condition makes it difficult to generate a phenotype–genotype pathway but case analyses can suggest an algorithm for genetic testing referral.

  • The increasing number of new referrals show raised awareness among UK paediatricians, but a centralised database is needed.

Introduction

Genetic disorders affecting normal metabolism and function of the pulmonary surfactant proteins have been recognised as causing respiratory problems in term neonates, children and adults. Professor Lawrence Nogee and colleagues were the first to describe these conditions and genetic mutations in Johns Hopkins Hospital in Baltimore.1–3 There are multiple mutations of the surfactant genes that cause overlapping phenotypes. Concomitantly, the pathophysiology involves not just deficiency of particular surfactant components (SP-B), but also dysfunction caused by aberrant protein accumulation (SP-C) and genetic defects in a specific transport protein (ATP-binding cassette subfamily A member 3; ABCA3). Therefore, the term surfactant dysfunction (SD) is now internationally used.1–3

Pulmonary surfactant is composed of lipid mixtures and specific proteins (surfactant proteins A, B, C and D: SP-A, SP-B, SP-C and SP-D). These surfactant proteins are synthesised and secreted by alveolar type II epithelial cells (AEC2). ABCA3 transports phospholipids essential for surfactant function into lamellar bodies. NKX2.1, the gene encoding for thyroid transcription factor-1, influences the expression of surfactant genes and is expressed in the lung, thyroid and central nervous system.

SP-B deficiency (MIM265120) was first described in 1993 in a term infant with diffuse lung disease when a frameshifting mutation of the surfactant protein B gene (SFTPB) was found (MIM178640). The disease is inherited as an autosomal recessive trait. The classical presentation is a term baby with clinical respiratory distress syndrome (RDS), unresponsive to therapy and with a typical radiological picture of hyaline membrane disease. Some patients have been placed on extracorporeal support for intractable respiratory failure and early death is common. There are reported cases of survival following lung transplantation in SP-B deficient children and cases of longer survival in partial deficiency caused by particular mutations.4 SP-C deficiency (MIM610913) was next described in 2001 in a term infant who developed respiratory symptoms and a radiological picture of interstitial lung disease (ILD) at 6 weeks and whose mother was in respiratory failure. The disease is caused by heterozygous mutation in SFTPC (MIM178620) and is inherited in an autosomal dominant fashion or may arise de novo. The age of onset and prognosis are highly variable with some cases being asymptomatic through adulthood. Lung disease caused by ABCA3 gene mutations was first described in 2004 in term babies with RDS and death in later infancy (MIM610921; gene601615). Further publications described cases of ABCA3 mutations that present as ILD in older children. The condition is autosomal recessive and there are mild cases when one mutation allows some ABCA3 function.5 Mutations of the NKX2.1 gene can cause RDS or ILD. Because the gene is expressed in the thyroid and central nervous system, patients with a mutation may have chorea or hypothyroidism, hence the term ‘Brain–Thyroid–Lung’ syndrome.6 There is no specific biomarker currently to diagnose SD, although elevated KL-6 levels (protein expressed by lung epithelial cells) have been observed and could distinguish from other diffuse lung disorders.7

There are multiple mutations known to cause surfactant disorders. At least 40 different mutations have been observed for SFTPB and SFTPC distinctively and over 150 mutations for ABCA3 gene. The frameshift mutation c.361delCinsGAA (previously called 121ins2) has accounted for 60%–70% of SFTPB mutations in the US population predicting an incidence for the disease of approximately 1 in 1.7 million births. A common mutation has also been found for SFTPC (c.218T>C (p.Ile73Thr)) and represents 25%–35% of all SP-C deficient cases. The ABCA3 mutation c.875A>T (p.Glu292Val) indicates a carrier rate of 1 in 275 individuals in the USA.1–3

Because the phenotypes are so similar, lung biopsy is performed in some cases. Histology under light microscopy is similar regardless of the genetic mutation and includes AEC2 hyperplasia, interstitial thickening with mesenchymal cells and macrophages and granular, eosinophilic proteinaceous material within the air spaces. Electron microscopy can distinguish in SP-B deficient cases that AEC2 do not contain normal lamellar bodies but organelles with variable sized vacuoles, while in ABCA3 cases lamellar bodies are replaced by the characteristic ‘fried-egg appearance’ of dense bodies.1

Up until 2006, UK children suspected of SD were diagnosed clinically and blood samples were sent to Professor Nogee in the USA for analysis. Following a Wellcome Trust Biomedical Research Collaboration Grant which funded two molecular genetics laboratory staff to familiarise themselves with surfactant gene mutation testing in Baltimore, the National Diagnostic Service was established at Great Ormond Street Hospital. Screening has now been offered since 2006, and the number of referrals is increasing every year (from 22 children in 2006 to 109 children in 2011). The cost of analysis is high (£1200 for all three genes), the test is time consuming (up to 2 months for the three whole gene screens and 2 weeks for carrier testing) and the condition is rare. This produces a low diagnostic yield and is therefore expensive as an exclusion test. Reasons for referral vary widely because clinical, imaging and histopathological features overlap. We therefore reviewed our positive results to see if we can identify clinical features that would lead to an improved referral algorithm.

Materials and methods

All cases referred for surfactant mutation screening from 2006 to 2011 to the North East Thames Regional Molecular Genetics Laboratory were retrospectively reviewed. They included children referred because of respiratory problems, in utero cases for antenatal diagnoses and parents of children with a molecularly confirmed diagnosis for carrier testing. Samples tested in overseas laboratories have not been included in this study.

The patients with a confirmed diagnosis by mutation analyses were grouped into SFTPB, SFTPC or ABCA3 mutations in a central database. For each individual case the referring consultant was contacted to provide more detailed information regarding clinical presentation, family history, investigations, medication and outcome in a standardised questionnaire.

Results

During the period 2006–2011, a total of 427 samples were received for surfactant mutation analyses from 15 paediatric centres. Out of these, 332 were from children as new referrals, 10 were prenatal diagnoses and 85 were parents for carrier testing and genetic advice.

There was an increase in the number of referrals each year while the number of positive cases remained low. The distribution of referrals and positive cases is illustrated in figure 1.

Figure 1

Distribution of referrals and confirmed positive cases each year.

Over the 6-year period we identified a total of 25 new cases positive for SD disorders (7.5% (25/332)). There were six new cases with SFTPB, seven children with SFTPC and 12 children with ABCA3 mutations. We also identified three SFTPB positive cases in fetuses as part of antenatal diagnosis.

Clinical presentation, family history, investigations, management, outcome and genetic results are presented in table 1 for SFTPB and SFTPC and in table 2 for ABCA3 positive cases.

Table 1

Characteristics for SFTPB and SFTPC cases

Table 2

Characteristic for ABCA3 positive cases

Respiratory distress at birth was the presenting symptom in all SFTPB and in 10 of 12 confirmed ABCA3 cases. SFTPC patients were more likely to present later with chronic cough, failure to thrive or oxygen dependency. While diagnosis in SFTPB was established soon after birth allowing withdrawal of ventilation in all cases with the longest survivor ventilated for 5 weeks, the diagnostic age varied from 3 months to 10 years in SFTPC and from neonatal period to 13 years in ABCA3 cases. The latest time for diagnosis in our cohort was at 13 years of age, although the patient presented with neonatal symptoms.

The outcome for cases with SFTPC and ABCA3 mutations varied. One SFTPC child died within the first year of life but the remaining six cases survived, the oldest being 10 years. Of the ABCA3 cases, five died early in infancy and seven survived. So, although a vast majority of ABCA3 cases referred to us for testing had onset of RDS at birth, more than half of this group managed to survive, with the oldest being 15 years.

Discussion

Since testing for surfactant protein mutations became available in the UK in 2006 there has been a considerable increase in the number of referrals from tertiary neonatal or paediatric respiratory units. Nevertheless, referrals seem to concentrate in a few centres suggesting that enthusiastic medical teams are sending more samples than necessary for testing, yet it is likely that the condition is underdiagnosed across the UK population. The vast majority of positive cases were term babies of more than 37 weeks gestation. However, we also encountered a 32-weeker who was homozygous for SFTPB mutation, had consanguineous parents and a family history of the condition. Gower and Nogee previously recommended testing from 38 weeks gestation or earlier in cases with positive family history.1 Our cohort would suggest testing from 37 weeks or as early as 32 weeks if parents are consanguineous or family members affected. Still, genetic testing should not be regarded as an exclusion test in premature babies with chronic lung disease.

It is universally recognised that SFTPB cases will present at birth with RDS, but we found respiratory distress was also the presentation for 10 out of 12 ABCA3 cases. This does not necessarily mean that RDS at birth is the most common presentation in ABCA3 patients but that patients who develop respiratory problems very early in life are more likely to be referred for genetic testing than older children with respiratory symptoms. Our cases with SFTPC dysfunction had onset of symptoms at birth or within the first months of life, and the ILD picture later in life was not the main clinical presentation. This implies again that late onset respiratory problems do not seem to trigger referral for genetic testing.

Interestingly, our series showed that all SFTPB cases had a positive family history although not all cases were from consanguineous parents. In the SFTPC group on the other hand, only a few had family history of respiratory problems suggesting many SFTPC individuals may remain untested and undiagnosed, probably because of mild presentation.

Ventilator dependence in a term baby was the main reason for referral in all SFTPB cases. However, the reason for referral in ABCA3 cases varied widely from RDS in a term baby with no response to surfactant to high oxygen requirement, unexplained ILD or suggestion of diagnosis after lung biopsy was performed. SFTPC children were referred for genetic testing due to a diagnosis of ILD made on chest CT and lung biopsy. These children underwent both chest CT and lung biopsy but were not referred for genetic testing until the biopsy results suggested an SD. As previously suggested,8 we would recommend that suspected cases should have genetic testing based on clinical symptoms, chest CT findings and family history before proceeding for lung biopsy as a less invasive approach. However, the mutation analyses take a few weeks and tend to be batched at present. Hopefully, new technical developments may shorten the time of processing genetic results in the future and this non-invasive diagnostic technique will precede invasive biopsies.

Genetic results showed a wide variety of mutations. For SFTPB children we detected the mutation c.195+5G>A in two families from different centres. It means that although SFTPB mutation c.361delCinsGAA (previously called 121ins2) is very common in the US population, in the UK children the mutation c.195+5G>A can be more often encountered. Genetic testing for SFTPC detected the commonly reported mutation, c.218T>C (p.Ile73Thr), in two unrelated cases presenting at 1 and 3 years with the children surviving beyond 3 and 4 years, respectively. The c.218T>C mutation therefore accounted for 2/7 (28.5%) of SFTPC alleles in our patients. The remaining five mutations seen were each reported in a single case. In ABCA3 cases, mutation c.875A>T (p.Glu292Val) was identified in different families from four medical centres. There are more than 150 mutations for ABCA3 described in the literature so far, but in our series c.875A>T was found in four cases with one child a homozygote (20.8% (5/24) ABCA3 alleles). Interestingly, children who have this mutation c.875A>T are all children who survived. Moreover, none of the children who died early in life had the mutation which makes us speculate this is probably a mild mutation.

The introduction of genetic testing for SD has brought a significant contribution to the diagnosis and genetic counselling in these rare entities. Moreover, if the screening for these genes detects no mutation, analysis for NKX2.1 deletions or mutations should be considered and the absence of neurological and thyroid signs should not preclude testing if other findings suggest SD.1 However, we need to recognise the limitation of testing in terms of the time required to perform the analysis and the existence of unknown mutations.9 Also, Peca et al published in 2011 a case of an infant with severe RDS at birth followed by recurrent respiratory failure episodes, hypopituitarism and neurological abnormalities where genetics identified a heterozygous ABCA3 mutation, but no SFTPB, SFTPC or NKX2.1 mutations or deletions were found.10 Immunofluorescence studies showed thyroid transcription factor-1 prevalently expressed in type II cell cytoplasm instead of nucleus, indicating defective nuclear targeting. Kinetic studies demonstrated a marked reduction of SP-B synthesis leading Peca et al to suggest that heterozygous ABCA3 missense mutations may act as disease modifiers in other genetic surfactant defects.

Conclusions and recommendations for testing

  1. Testing for surfactant dysfunction should be considered in term neonates more than 37 weeks gestation who develop unexplained respiratory distress. Testing can also be considered in earlier gestational age when there is a positive family history and consanguinity, but not as a routine exclusion test in preterm babies with chronic lung disease.

  2. The initial testing in neonatal period should be for surfactant protein B and ABCA3 but if respiratory distress persists and defines as interstitial lung disease then SFTPC testing is justified.

  3. In cases with clinical and radiological picture of interstitial lung disorder and late onset, consider genetic testing for SFTPC and ABCA3 before lung biopsy especially when the family history is positive.

  4. It is important to accept the limitations of genetic testing as the technique could take weeks and mutations outside the coding region that affect gene expression may not be detected.

Acknowledgments

We would like to thank all patients and their families and all the clinicians who contributed to the data collection: C Wallis, Great Ormond Street Hospital, J Davies, Royal Brompton Hospital, T Watts and A Kaiser, Evelina Children Hospital, K Luyt, St Michael's Hospital Bristol, C Murray and F Child, Royal Manchester Children Hospital, G Connett, Southampton University Hospital, D Lacy, Wirral University Teaching Hospital, S Ellis and K Blake University Hospitals of Coventry, M Desai, Birmingham Children's Hospital, S Mayell, Alder Hey Children Hospital, and R Coombs and N Jay, Sheffield Children Hospital.

References

View Abstract

Footnotes

  • Contributors QM conceived and coordinated the study. ST and AG coordinated data collection. ST, AG and QM produced the manuscript. EA is responsible for the molecular genetic surfactant service including interpretation and reporting of the results. LJ is head of the Molecular Genetics service and provided critical evaluation of the manuscript.

  • Funding Genetic testing was made possible by a Wellcome Trust Biomedical Research Collaboration Grant (ref 056728/Z/99/Z) to Quen Mok.

  • Competing interests None.

  • Ethics approval Project approved by the National Research Ethics Service Committee London- Bloomsbury.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data sharing statement The data have not been previously shared.