The need to assess the pathogeneticity of genetic variants

Genetic sequencing is increasing diagnosis of pre-existing conditions, identifying which diseases a patient is at risk of developing, and which treatments they will best respond to. As the cost of genetic sequencing falls its use in clinical, research and private testing will continue to increase. Whilst we know that variation is a natural feature of the human genome, we are currently unable to accurately predict the effect on health, and the penetrance of this effect, for the majority of reported variants.

The variable pathogenicity of putative Loss-of-Function (pLOF) mutations was the focus of a recent paper by Johnston et al., (2015): genetic sequencing identified 103 individuals (from 951) carrying a pLOF mutation in a gene previously associated with haploinsufficiency pathologies. Of the 79 patients available for in depth clinical follow-up screening 43% had an individual or family history attributable to the variation. This included two undiagnosed BHD patients who can now be monitored for the development of renal cancer. However, 54% of these patients were positive for a pLOF variant but had no clinical indicators. This included a family carrying a mutation in the X-linked DMD gene, the cause of Duchenne muscular dystrophy, but with no evidence of muscle weakness in either male carrier. It is unknown whether these pLOF mutations are non-pathogenic variants or have variable penetrance. Potentially such patients could help identification modifier genes and increase understanding of disease pathology.

Determining whether a variant is non-pathogenic or non-penetrative is important both when a mutation is being sought and for incidental findings resulting from a more general genetic analysis. Patients should be warned in advance that mutations in disease-risk genes could be identified as a result of testing. The ACMG provides recommendations on reporting incidental findings (Green et al., 2013) but the variable penetrance of even well-known cancer-risk genes such as BRCA1 (Petrucelli et al., 2013) can make calculating risk complicated.

This difficulty in assessing the risk factors associated with genetic variants was highlighted in a recent review by ClinGen which reported variation in the interpretation of 17% of variants identified in more than one genetic lab (Rehm et al., 2015). These interpretations, stating either that a variation is non-pathogenic or highly pathogenic, could influence and potentially compromise patient care. Therefore it is essential that more accurate assessments of risk can be conducted.

The NCBI hosts a genetic variation database, ClinVar, which encourages private, clinical and research labs to submit genetic testing data which is then accessible to the wider research community (coded for anonymity). Currently over 300 different labs worldwide contribute data and over 172,000 variants in 23,000 genes have been reported. ClinGen are using this database to assess the clinical relevance of genomic variants – a large project as 71% of known variants are of “uncertain clinical significance” and even the majority of the 29% “likely or known to be pathogenic” have only been reported once (Rehm et al., 2015). The submission of further genetic data, both to general and disease-specific databases such as the LOVD-hosted FLCN mutation database, will enable researchers to make more robust assessments of individual variant pathogenicity.

Large scale sequencing projects such as the WGS500 and 100,000 Genomes Project can discover more about rare disease and cancer patients’ genomes, and potentially increase understanding of pathology (Taylor et al., 2015). However, to be able to accurately assess the risk factor of disease variants similar large sequencing projects in reportedly healthy individuals are required to identify variants that are common in the population and therefore most likely non-pathogenic. For now, even if variant risk cannot be accurately calculated, genetic sequencing enables identification of suspected pathogenic variants, which provides the basis for further clinical evaluation and care of patients and their families.

 

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Advancing rare disease treatments: patient groups and industry

Rare diseases have become a more interesting target to the pharmaceutical industry as a result of incentives offered by the USA Orphan Drug Act (1983) and EU/141/2000 legislation, the increasing development costs and failure rate of common disease drugs, and the potential for rare disease drugs to influence common disease treatment. It is predicted that revenue from rare disease drugs will continue to rise and that more pharmaceutical companies will divert resources towards their development (Orphan Drug Report 2014). However, the traditional drug development methods are not optimal for rare diseases as experts and patients tend to be sparse. Arguably the best way around this issue is to collaborate with a patient group – the concept of last week’s Findacure workshop in London.

Patient groups are experts in the disease, know the expert clinicians, the researchers and, more importantly, the patients. They can help design more accessible and suitable clinical trials (see our previous blog on clinical trials for rare disease patients), and can facilitate more long-term patient follow-up. Collaborations are also beneficial to the patient groups as there is a focus on their disease, increasing awareness and knowledge, and their members gain access new clinical trials.

However, such collaborations are often scrutinised by external groups and accusations of undue influence can be damaging to the patient group who must maintain the trust of their patients and partners. As collaborations have such scope for treatment development, it is important that they be protected from such attacks and that all parties are comfortable with continued interactions. For this reason it is essential that collaborations have predefined rules of engagement, maintain transparency for all interactions, especially those with a financial incentive, and welcome external scrutiny and assessment. In addition the majority of pharmaceutical companies will have strict codes of conduct, often including regulations regarding patient group contact. It is important however that both parties should be involved in drafting a collaborative agreement ensuring that both group agendas are fairly represented.

Patient groups should play an active role in these collaborations; acting as a voice for their patients, chasing unanswered questions and ensuring data dissemination. However, for patient groups to ask the right kinds of questions and have a meaningful input, industrial partners must ensure they provide understandable information about ongoing work and results. A recent survey found that only 22% of the general public in several European countries were knowledgeable about drug discovery and clinical trials (EUPATI Public views on Medicine Development). To increase understanding among the public, and importantly within patient groups, organisations such as EUPATI provide training, workshops, webinars and online resource libraries to help in the development of the skills required to form mutually beneficially collaborations.

The power of patient groups to impact the development of rare disease treatments is likely to increase following the recent launch of Rare Disease International (RDI) – a global organisation advocating for recognition of rare diseases as an international public health and research priority, thereby increasing the services and support available to patients and their families. RDI aim to facilitate networking, increase education of rare diseases on a national and international level, and increase rare diseases representation in the pharmaceutical industry. They will also be able to offer support and guidance to individual patient groups considering collaborations.

However, one of the largest issues associated with the development of treatments for rare diseases cannot be influenced by these groups alone: the cost of treatments. Pharmaceutical companies must recuperate the costs associated with the R&D and clinical trials for any new treatments. Unfortunately the limited number of target patients for any single rare disease most often results in these costs being extremely high per patient, severely limiting access. It is only through an overhaul of the pricing structures, and questioning profit after recuperation, that this issue can be addresses and newly developed treatments can be accessed by those most in need.

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A lactate-induced response to hypoxia

Hypoxia regulation ensures cell survival and growth in low oxygen environments. HIF signalling is a well-established element of this regulation but is also associated with tumourigenesis in BHD, VHL, HLRCC, TSC, and sporadic cancers. New research from Lee et al., (2015) has identified a second, HIF-independent, hypoxia response which can modify cell survival and growth signalling pathways – the lactate-induced activation of NDRG3-mediated signalling.

Oxygen-dependent hydroxylation of HIF-1α and HIF-2α by prolyl hydroxylase domain (PHD) enzymes and ubiquitination by pVHL results in proteosomal degradation inhibiting HIF signalling. Immunoprecipitation identified NDRG3, a protein previously implicated in cell proliferation and migration signalling (Melotte et al., 2010), as also interacting with PHD2. Knockdown of either PHD2 or pVHL increased NDRG3 protein levels, indicating normoxic post-translational degradation by PHD2/VHL (Lee et al., 2015).

NDRG3 protein levels increased in multiple cell types in response to hypoxia and were correlated with increased angiogenesis, anti-apoptotic, motility and proliferative (but not metabolic) gene expression. A critical role in these processes was confirmed when reduction in NDRG3 abolished the hypoxia-induced expression of pro-angiogenic factors, increased cellular apoptosis under prolonged hypoxia and decreased tumour cell growth.

NDRG3-accumulation in hypoxia occurs after HIF activation and is correlated with HIF-1α-mediated cellular lactate production; NDRG3 activity is not directly HIF-dependent but is coupled to the HIF-mediated hypoxia response. Lee et al. determined that lactate physically binds to NDRG3 blocking VHL-mediated ubiquitination and proteosomal degradation. Further work is required to determine if lactate is also inhibiting the PHD2-mediated hydroxylation required for VHL binding.

NDRG3 increased phosphorylation of c-Raf and B-RAF suggesting a role in activation of the Raf-ERK signalling pathway – associated with promoting cell growth and increased expression of angiogenic markers in hypoxia and tumours (Roberts & Der, 2007). Lee et al., propose two phases in prolonged hypoxia that enable cells to cope: initially HIF-mediated changes to metabolism increase the production of biosynthetic building blocks and results in lactate build-up, then NDRG3-mediated signalling pathways provide cues for cellular growth and angiogenesis.

However, this self-sufficient mechanism that enables recovery from hypoxia, can also be used by tumour cells enabling continued growth. Lee et al., reported increased levels of NDRG3 and ERK1/2 signalling molecules in engrafted tumours and patient hepatocellular carcinoma samples (n=25/103) supporting a role in tumour development. Given the importance of pVHL in NDRG3 regulation, the NDRG3-Raf-ERK signalling pathway could also be playing a role in VHL-associated tumourigenesis.

Cancer cells commonly show metabolic changes and increased dependency on glycolysis leading to increased lactate production. Lactate can be an alternative energy source and an inducer of tumour angiogenesis (Doherty & Cleveland, 2013), but lactate dehydrogenase inhibition can suppress tumour growth (Le et al., 2010). Determining NDRG3 as a mediator of lactate-induced responses has identified a new therapeutic target; targeting NDRG3 in combination with HIF signalling could increase treatment efficiency by reducing apoptotic-escape via a HIF-independent pathway (Lee et al., 2015).

NDRG3 targeting treatments or combination treatments could prove effective for BHD as a loss of FLCN has been linked to increased lactate production (Preston et al., 2011) and ERK1/2 signalling (Baba et al., 2008, Hudon et al., 2010). In addition, impairing lactate production in UOK257 (FLCN-null) cells reduces cell growth (Preston et al., 2011) which could indicate an important role for NDRG3-Raf-ERK signalling in BHD tumourigenesis. Treatments that either directly target NDRG3 or that block lactate accumulation could therefore be beneficial in the treatment of a wide range of cancers.

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HIF-2α inhibition rescues zebrafish VHL model phenotypes

Patients with VHL develop a range of hypervascular tumours including renal cell carcinoma (RCC), retinal and central nervous system hemangioblastomas (HB). The loss of VHL protein (pVHL) reduces HIF protein degradation increasing HIF-1α and HIF-2α signalling and the expression of target genes involved in angiogenesis, erythropoiesis, metabolism and cell proliferation. Increased HIF signalling has also been linked to tumourigenesis in sporadic RCC, BHD, TSC and HLRCC (Kim et al., 2006, Preston et al., 2011). HIF-2α is reported to be more important than HIF-1α in renal tumourigenesis – specific inhibition of HIF-2α has been shown to be sufficient to block aberrant growth in VHL-null cell lines (Kondo et al., 2003, Zimmer et al., 2004). As such the development of treatments that target HIF-2α may be more therapeutically beneficial that current downstream targeted therapies which show little efficacy in VHL patients.

Metelo et al., (2015) report that HIF-2α-specific inhibition can reverse vhl-associated pathologies in a zebrafish model – a good model system for VHL as the hypoxia, angiogenesis and erythropoiesis constitute pathways are conserved between mammals and fish. The authors assessed the ability of compound 76, originally identified in a cell line (786-O) screen (Zimmer et al., 2008), to reduce HIF-2α signalling both in the vhl-/- model zebrafish and wildtype zebrafish that were hypoxia-challenged using the hypoxia-mimetic DMOG. In both groups, when untreated, there was an increase in vascular branching – most notable in the trunk and head – and erythrocytosis. The vhl-/- zebrafish also have cardiomegaly with decreased cardiac contractility believed to be the major cause of death during early development.

Compound 76 inhibits HIF-2α by promoting iron regulatory protein 1 (IRP1) binding to 5’-UTR of HIF2a-mRNA thereby blocking translation (Zimmer et al., 2008). As HIF-1α mRNA does not have a IRP1-binding loop inhibition is specific to HIF-2α. Metelo et al. used qRT-PCR to determine levels of HIF1α and HIF-2α target genes in hypoxia-challenged zebrafish to confirm inhibitor efficacy and specificity was retained.

Incubation of hypoxia-challenged or vhl-/- zebrafish with compound 76 reduced vascular sprouting in the trunk and brain. As the complex networks formed in the nervous system are reminiscent of the highly vascularised HBs in patients this suggests the HIF-2α inhibition treatment could be effective for multiple VHL pathologies. The compound 76 treated zebrafish also showed decreased erythrocytosis potentially through increased terminal differentiation to mature erythrocytes. Compound 76 also increased cardiac contractility in vhl-/- zebrafish, which is most likely responsible for the reported increase in early larval survival.

The efficacy of compound 76, a non-optimised compound, in a vertebrate VHL model raises hope for its eventual use in patients, although further refinement is required before use in clinical trials. PT2385, an inhibitor which binds to the HIF-2α protein blocking the heterodimerisation required for target gene transcription (Scheuermann et al., 2013), is currently in phase I clinical trials for advanced clear cell RCC (NCT02293980). Preclinical trial data suggests that PT2385 is a potent and selective inhibitor that can readily be absorbed from oral preparations (Peloton Therapeutics).

In BHD increased HIF signalling is the result of aberrant activation of AMPK and increased PCG1α activity initiating mitochondrial biogenesis and ROS production (Yan et al., 2014). Increased expression of HIF-2α was reported in BHD pulmonary cyst samples (Nishii et al., 2013) and although the authors did not assess levels of HIF-2a the increase in VEGF in these samples would suggest increased HIF-2α expression. If HIF signalling is associated with multiple aspects of BHD pathology then HIF-2α inhibitors could prove effective treatments for multiple currently untreatable rare diseases.

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The potential use of BH3-mimetics to overcome apoptosis-resistance in renal cell carcinoma

Renal cell carcinoma (RCC), as well as other solid tumours, can prove resistant to standard cancer treatments such as chemotherapy. One mechanism likely to play a role in this resistance is activation of HIF signalling either as a result of hypoxia within the tumour or as the result of mutations as in BHD, VHL and TSC. Increased HIF activity, as well as altered PI3K/AKT/mTOR, MEK/ERK and TGFβ signalling, can shift the balance of anti- and pro-apoptotic factors enabling tumour cells to survive in unfavourable conditions.

In response to cell death signals BH3-only family members, such as BIM, bind to and sequester the anti-apoptotic BCL-2 family members, liberating the pro-apoptotic BAX and BAK proteins that induce apoptosis (Figure 1). However, tumour-specific loss of BIM, and associated resistance to apoptosis, has been reported in several tumour types: in clear cell RCC samples (35/45) with a correlation between BIM expression and apoptosis-susceptibility identified in RCC lines (Zantl et al., 2007); in BHD tumour samples (Cash et al., 2011) and VHL-deficient cell lines (Guo et al., 2009) suggesting a role for FLCN and pVHL in BIM expression and stability respectively; and in breast cancer tumour lines with aberrant PI3K/AKT/mTOR or MEK/ERK signalling (Faber et al., 2011).

Figure 1

Figure 1: BIM binds to and sequesters Bcl-2 and Mcl-1 to induce apoptosis via dimerisation of BAX and BAK.

One potential treatment for such tumours is the use of BH3-mimetics – small molecule inhibitors, such as ABT-737 (or the orally bioavailable formulation ABT-263) and Obatoclax (OBX), which bind to BCL-2 and induce apoptosis (Figure 2, Oltersdorf et al., 2005, Tse et al., 2008, Nguyen et al., 2007). OBX has a broader specificity that ABT-737 binding MCL-1 as well as BCL-2, making it a more potent antagonist. Additionally it has been shown that OBX can reduce mTOR activity in melanoma cells (Espona-Fiedler et al., 2012) and both disrupt HIF-1-α protein synthesis and enhance proteosomal degradation to lower HIF-1α protein levels under hypoxic conditions (Gariboldi et al., 2015). As HIF signalling is often perturbed in RCC this suggests that the use of BH3-mimetic may also be useful in these cases.

Figure 2

Figure 2: BH3-mimetics can bind Bcl-2 and Mcl-1 to induce apoptosis in the absence of BIM.

 

BH3-mimetics could be of particular interest in BHD as several of the pathways known to be altered in FLCN-deficient cells have been implicated in the control of BIM expression: increased HIF-signalling plays a role in BHD renal and pulmonary pathology (Preston et al., 2011, Nishii et al., 2013); increased mTOR activity has been seen in patient samples and BHD models (Baba et al., 2008, Nishii et al., 2013); and increased activation of ERK1/2 signalling was identified in FLCN-null kidneys (Baba et al., 2008).

In addition to these pathways Cash et al., (2011) reported a TGFβ-dependent reduction in BIM expression and increased apoptotic resistance in Flcn-/- ES cells, with Flcn-restored cells showing rescued apoptotic response. A role for FLCN in the regulation of TGFβ signalling had previous been suggested (Hong et al., 2010) following the reduction in TGFβ-target expression in FLCN-null cells (including SMAD7 as discussed in a previous blog regarding a role of MMPs in BHD). Cash et al. determined that the deregulation of TGFβ signalling resulted in hypoacetylation of target promotors, including BIM, and reduced expression. Treatment of these cells with either ABT-737 or an HDAC inhibitor increased susceptibility to apoptosis identifying them as potential therapies in BHD.

BH3-mimetics have proven successful in a range of cancers and there are several ongoing trials assessing the efficiency of ABT-263 and OBX in the treatment of solid tumours. The reduction in BIM expression in BHD tumours and the links between FLCN-associated signalling and BIM expression suggest that BH3-mimetics could be a valid treatment in BHD. Interestingly Cash et al. reported a reduction in BIM levels in a fibrofolliculoma sample raising the possibility for BH3-mimetics to be used for multiple BHD phenotypes.

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Clinical trials for rare diseases – finding and keeping patients

International Clinical Trial day (May 20th) celebrates the medical advances as a result of clinical trials. Clinical trials are essential to ensure drug safety and efficacy, and the recent increase in the development of orphan drugs has led to an increase in rare disease clinical trials. The nature of rare diseases creates specific challenges for clinical trial design and patient recruitment.

A major challenge in any clinical trial is the recruitment of enough suitable patients – something made more difficult with a rare disease because there are so few patients meaning recruitment has to occur from a wide geographical area. Patient support groups are key in identifying and contacting these patients, and often know of equivalent groups in other countries. Increasingly they have patient registers that can contain information on patient location, genetics, health and sometimes biological sample data. Early contact with a patient support groups can also help identify clinical or research experts and current treatment centres (and their patients) that could assist with a trial. Additionally a dedicated trial website, suitably advertised, enables patients to learn more about the trial independently and enquire if interested.

A good trial design ensures valid and useful results however the “gold-standard” of a randomised, parallel group controlled trial may not be feasible with rare diseases due to low patient numbers and a lack of comparative interventions. Instead rare disease trials are more likely to have under 50 patients and be single arm, non-randomised, open label trials (Bell & Tudar Smith 2014). Details of the different approaches for designing an effective clinical trial design for rare disease can be found in a methodical review from Gagne et al., (2014).

Rare disease clinical trial logistics also require additional thought and a more patient-centric approach can ensure adequate recruitment and retention. Early consideration of acceptable physical burden, treatment durations and travel requirements, which support groups can often advise on, can speed up patient recruitment.

Many rare diseases are debilitating and/or affect children so patients will require an accompanying carer for visits – which increases travel and accommodation costs. Multiple trial centres, potentially across several countries, can help reduce the required travel and be favourable to patients. An example is the AKU Society developAKUre trial which has trial centres in the UK, France and Slovakia enabling more local participation. These countries were chosen based on patient support groups and registries, clinical and research expertise and in Slovakia a higher than average patient density. AKU patients from throughout Europe are also travelling to the UK to participate which requires additional planning and patient support such as translation services.

An alternative clinical trial strategy, which takes patient preference and potential difficulty travelling into account, is the provision of an in-home clinical service; the majority of simple clinical procedures (treatment administration, blood draws, health monitoring) are administered by a clinical nurse at home with travel to the designated clinical trial site only required for certain appointments. This significantly reduces the disruption to patients and their families in terms of time and can reduce travel and accommodation costs for the sponsor. In-home supported clinical trials have been found to have reduced withdrawal rates and the reduced burden on patients can encourage greater enrolment and completion of trials ahead of estimates (Norris et al., 2012).

Choosing to take part in a clinical trial is a personal decision and a patient has the right to withdraw at any time. By supporting rare disease patients from the start and throughout, making taking part in clinical trials as easy as possible, more effective treatments for a range of rare diseases can hopefully be approved for use. Information about ongoing trials can be found at clinicaltrials.gov. Participation in a clinical trials is not entirely risk free and should always be discussed with your doctor. You can find more information about the process of clinical trials here.

  • Bell SA, Tudur Smith C. A comparison of interventional clinical trials in rare versus non-rare diseases: an analysis of ClinicalTrials.gov. Orphanet J Rare Dis. 2014 Nov 26;9:170. PubMed PMID: 25427578.
  • Gagne JJ, Thompson L, O’Keefe K, Kesselheim AS. Innovative research methods for studying treatments for rare diseases: methodological review. BMJ. 2014 Nov 24;349:g6802. Review. PubMed PMID: 25422272.
  • Norris N, Pascale W, Tulipano D. In-home Clinical Services: Reducing patient burden and improving patient participation in studies. ACRP Clinical Research Articles. April 2012. http://www.symphonyclinicalresearch.com/Final%20ACRP-Publication.pdf

Thank you to Oliver Timmis at the AKU Society for providing insight into the developAKUre trial.

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A role for Matrix Metalloproteinases in BHD?

The BHD protein folliculin (FLCN) plays a role in numerous signalling pathways and cellular processes. Although mutations in FLCN are only firmly linked to the development of fibrofolliculomas, pulmonary cysts and renal tumours it is possible that disruption of these pathways also plays a role in other phenotypes. Recently Kapoor et al., (2015) reported three cases studies of women with BHD who presented with intracranial vascular pathologies. There are few other reports of vascular pathologies in BHD and further studies would be required to determine any causative link. Kapoor et al. proposed two hypotheses to link BHD to aneurysms and vascular malformations: aberrant HIF-1α signalling and increased matrix metalloproteinase 9 (MMP-9) activity.

MMP-9 is a proteolytic enzyme that cleaves several connective tissue proteins and has multiple roles in tissue extra-cellular matrix (ECM) remodelling, angiogenesis and cell migration. Regulation of MMP-9 is predominantly transcriptional; low background levels result from inhibition by SMAD7 (Kim et al., 2012, Yu et al., 2013). A significant reduction in SMAD7 expression, alongside other TGF-β signalling targets, was reported in FLCN-null cells (Hong et al., 2010), supporting a link between reduced FLCN and increased MMP-9.

MMPs are important for vascular wall matrix remodelling and increased activity is associated with potentially-pathogenic increased degradation of structural proteins in vascular walls (Maradni et al., 2013). Increased levels of MMP-9 have been seen in extra- and intra-cerebral, abdominal aortic and intracranial aneurysm walls (Pannu et al., 2006, Maradni et al., 2013). The expression of MMP9 is locally, rather than systemically, perturbed in such patients as healthy vascular tissue from aneurysm patients shows no increase in MMP-9 compared to healthy controls (Kim et al., 1997). Although an increase in MMP-9 has not been investigated in aneurysm wall samples from BHD patients, a role for FLCN in the control of MMP9 expression could support the potential for increased risk of intracranial vascular pathologies in BHD.

Aberrant MMP-9 activity has also been associated with a number of pulmonary disorders and previously been discussed in the context of BHD. MMP9 is expressed at low levels in healthy adult lung tissue but marked increases have been reported in cystic fibrosis (Sagel et al., 2005), asthma, IPF, and COPD (Atkinson & Senior, 2003) associated with airway and vascular remodelling. Increased MMP-9 expression is also seen in Lymphangioleiomyomatosis (LAM) patients associated with ECM breakdown and cystic lesions (McCormack, 2008).

In BHD patients increased MMP9 expression has been reported in alveolar epithelial cells, macrophages and neutrophils (Hayashi et al., 2010, Pimenta et al., 2012). However, these reports are not conclusive as there was a lack of comparison to healthy tissue (Hayashi et al., 2010) or no published genetic confirmation of BHD (Pimenta et al., 2012). Additionally increased production of MMP-9 as a result of increased pulmonary inflammation has been reported in mouse models of BHD (Goncharova et al., 2014). Contrary to these reports Nishii et al., (2013) found that MMP-9 levels were unchanged in their patient’s lung samples suggesting that further, large-scale analysis of expression is required in BHD patients.

The patient reported by Pimenta et al. (2012) was initially misdiagnosed with LAM and received standard treatment with the MMP-inhibitor doxycycline. Although this patient was subsequently suspected to have BHD she did show an improvement in pulmonary function during the treatment. Further research is required to understand the role, if any, that MMP-9 or other MMPs play in the pathology of BHD. If disruption of TGF-β signalling is resulting in a pathogenic activation of MMP-9 then it may be possible to utilise the well-established inhibitors such as doxycycline for the treatment of BHD pathologies.

 

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