Dr Hector G Lopez-Ruiz
email: hlopezruiz@gmail.com

PASSENGER MOBILITY AND CLIMATE CONSTRAINTS: ANALYSING ADAPTIVE STRATEGIES

Keywords: Greenhouse gas, long term, scenario, transport, sustainable development

Today, numerous works conclude that transport seems to be completely coupled to economic growth. Therefore, as a direct consequence of economic development, transport sits today as one of the major final energy consumers and one of the most important sources of carbon dioxide emissions. Furthermore, in the absence of major technological change, this unsustainable situation will most undoubtedly get worse in the future.

In this paper we analyze what different types of public policies aiming at sharp reductions in GHG emissions imply on passenger transport and how they can be linked to new behavior patterns affecting time use and consumption. For this, we use the TILT (Transport Issues in the Long Term) model's core microeconomic choice model IT-UP (Integrated Tools for Utility-based Planning). Through this analysis, we explain the interest of adaptive strategies for GHG mitigation.

I. INTRODUCTION

Today, numerous works conclude that transport seems to be completely coupled to economic growth. Therefore, as a direct consequence of economic development, transport sits today as one of the major final energy consumers and one of the most important sources of greenhouse gas emissions (GHG). Furthermore, in the absence of major technological change, this unsustainable situation will most undoubtedly get worse in the future.

Furthermore, recent scientific insight has shown that in order to reduce the climate change risk (overshooting a 2°C increase in global temperatures), global emissions should be cut by at least 50 % in the next 40 years. Consequently, for developing and developed economies to be able to attain the 50% mark, industrialized countries must reduce their emissions by at least 75%. This is not an easy task, in order to plan for these drastic reductions, numerous studies (BANISTER & HICKMAN; KATO & ITO et & LUTSEY) have looked into different options on how to get to this desired future. These studies concur on the fact that new technologies and their widespread use will be necessary in order to attain considerable GHG reductions, but they also agree that these new technologies will not be enough for industrialized countries to get to their objectives. Indeed, most works conclude that it would also be necessary to increase the match between new technology supply and consumer demand through the use of incentive economic instruments.

In this manner, GHG mitigation strategies imply the need to set up a certain number of public policies ranging from inciting technological progress, to tolls, to intermodal development or even rationing (tradable emission permits). Currently, an increasing number of countries have started to set up different types of programs to try to influence behavior in passenger mobility (especially personal vehicle mobility) and curb emissions from the transport sector, but are these initiatives enough?

This paper has three aims: Firstly, we explore -on the basis of long-term scenarios for the French economy-how a continued trend in the vehicle market (although promising concerning GHG reductions because of lower consumption factors) is not viable in the long-term future from an infrastructure point of view. Secondly, we offer insight on how requiring changes in behavior through public policies can offer more viable solutions for mitigation of GHG emissions in developed economies. Lastly, we will explore how policies aiming at mitigation of emissions can cause imbalances on a microeconomic level which lead to envisaging adaptive strategies in order to increase policy acceptability.

In order to better explain how we have carried out our analysis, we will present the reader with a brief description of the overall inner workings of the model used for this paper (TILT) while focusing particularly on the microeconomic module (IT-UP) which will be used extensively throughout the paper.

II. THE TILT MODEL (TRANSPORT ISSUES IN THE LONG TERM)

The TILT model has been designed to be a long-term equilibrium model by combining a macroeconomic and a microeconomic structure in a backcasting approach that takes into account new motor technologies and facilitates sensitivity and impact assessments through five modules that work on three different geographical scales (urban, regional and interregional) :



a macroeconomic model based on a re-foundation of the energy-environment modeling structures in order to properly assess long-term modifications of demographic variables and their impact on economic productivity and time use. This module was adapted from the BASES module in the VLEEM model (Consortium VLEEM, 2002),  a microeconomic model based on discrete choice modeling -adapted from ant algorithms-(LOPEZ-RUIZ, 2009) that takes into account transport cost, infrastructure capacity and quality of service,  a vehicle fleet dynamic model that analyses technological impact based on market penetration probabilities for new motor technologies and vehicles' survival rates,  a public policy model that joins a sensitivity analysis and a multicriteria analysis in order to offer a detailed assessment of the effects of different public policies on oil consumption and GHG,  an impact assessment model based on an input-output equilibrium analysis that details impacts on employment and production by sector.

The TILT model is centered on the simple idea of defined behavior types -in which speed/GDP elasticities play a key role-in order to determine macroeconomic transport demand estimations. The TILT model supposes that modal split in transport is directly linked to the idea that modal speed; transport times, transport management and household/firm locations determine modal shares. In this manner, the model's main hypothesis is that transport modal saturation rhythms can be varied -in the model-through public policies that have an effect on household/firm location and speed/GDP elasticities [START_REF] Crozet | Comment satisfaire les objectifs internationaux de la France en termes d'émissions de gaz à effet de serre et de pollution transfrontières? Programme de recherche consacré à la construction de scénarios de mobilité durable[END_REF][START_REF] Lopez-Ruiz H | Environnement & Mobilité 2050: des scenarios pour le facteur 4 (-75% de GHG en 2050[END_REF].

Furthermore, the model is also able to asses the system's sensitivity to public policies, investment needs in infrastructure and economic impact of different public policies whilst taking into account microeconomic choices. In this manner the TILT model structure enables the user to calculate energy consumption and pollutants emitted by transport activity (freight and passengers) on different geographical scales based on behavior patterns that can be influenced by public policies. In sum, the model has three main functions:



modeling passenger-kilometers and ton-kilometers coherent with a micro/macro equilibrium structure according to motor technology used for journeys and area of service,



modeling the vehicle park according to: age; motor technology; and year of production (for freight and passengers),



modeling and assessing public policy impacts on CO 2 emissions, infrastructure investment needs as well as overall impact on the economy.

By joining these three functions of the different TILT modules, it is possible to build scenarios that:



quantify the consequences of transport on the environment whilst detailing the systems' structure according to behavior and organizational changes and motor technology,



give a precise view of traffic by motor technology, gas consumption and emission levels for each type of transport according to service distances, type of vehicle and transport cost,



assess impacts of different policy pathways according to different scenario configurations.

These results, coupled with the model's structure, make TILT a powerful tool for building and exploring scenarios. The utility of the TILT model lays not only in its capacity to be flexible concerning different transport policies, changes in demography, behavioral differences as well as changes in transport structure and cost but also in its capacity to integrate a microeconomic insight module, on which this paper will be focused.

The TILT microeconomic sub-model lets us understand how, according to past tendencies (characterized by the coupling between growth and mobility), future public policies will impact demand for transport services as well as trade-offs linked to behavioral change and infrastructure use on different geographical scales.

IT-UP is largely inspired by developments done on ant algorithms (DORIGO, M. Di CARO, G. GAMBARDELLA, L.M, 1999) and their application to freight and passenger transport [START_REF] Lopez-Ruiz H | Environnement & Mobilité 2050: des scenarios pour le facteur 4 (-75% de GHG en 2050[END_REF]). This model relies on the idea of a representative agent that optimizes its transport decisions by taking into account opportunity (defined as the sum of goods and services that can be consumed in a period of time, LINDER, S. 1970) and cost in respect to a certain level of service on infrastructure -measured through a lateness index (LOMAX. T. TURNER. S, 1997).

IT-UP considers that the lateness index is defined by the difference existing between normal transit time and real transit time. This last indicator is useful in factoring in speed, distance and time into the calculation of the choice model and has the convenience of being comparable between modes.

In this manner, the proposed framework lets us asses the representative agent's choices that are coherent with the transport structure and its level of service. In the model, the value assigned to each choice (aij(t) -which refers to the choice of mode used to move from point i to point j) is calculated using the following equations:

(1) 1Where:

        i Ni ij N j t t t a              ) ( ) ( ) (
(2)

t time acces time services goods cos * ) ( &     and (3) ) ( ) ( t index Lateness t  
The inherent logic of the microeconomic module is particularly useful in technicoorganizational public policy assessments as it enables an analysis based on the idea that public policies are implemented as increasing/decreasing constraints on the system, in view of getting to a certain objective. Consequently, this facilitates the building of scenarios where a wide variety of social effects on different levels and aspects are comprised. In this manner, the TILT model is capable of giving insight on how changes in the transport structure linked to environmentally oriented public policies might influence passenger behavior in the future.

The following paragraphs will show how the theoretical framework of the IT-UP model can assess social effects on a public level for new behavior patterns that will undoubtedly need to be accompanied by clearly defined adaptive strategies.

III. BAU SCENARIO OVERVIEW, CAR MARKET TRENDS AND INFRASTRUCTURE INVESTMENT ASSESSMENT

In 2008, the TILT model was used to develop three technico-organizational scenarios in order to quantify the effects of climate oriented policies in the transport sector (LET-ENERDATA, 2008. The original version of the report is in French, a detailed description in English, can be found in LOPEZ-RUIZ & CROZET, 2010). The main aim of these scenarios was to test the efficiency of public policies (modeled as growing constraintsranging from promoting new motor technologies to public policies aiming at multimodality and decoupling transport activities from GDP) on GHG emissions.

In this study, the underlying principle of incremental constraints on the system allowed to present three different scenarios that allow a quick comprehension of the GHG reductions that can be obtained through policy mixes. Consequently, the scenarios offer a good representation of the general policy pathways usually accepted as being efficient and viable options for long-term oil consumption reductions. On this basis we will develop how the presented microeconomic framework can help in the analysis for each scenario and can give insight on adaptive strategies for sustainable planning.

The 2008 LET-ENERDATA report assessed sustainable transport scenarios for the French economy with a specified objective of -75% in GHG by 2050 through the identification of the different equilibriums possible that allow the attainment of the specified future. From these possible equilibriums, the three that best depict the range of solutions available -through public policy-were chosen:

• promoting strict technology standards -business as usual (BAU),

• green multimodality,

• decoupling transport activities from economic growth (GDP).

The results for each scenario were obtained by modeling a mix of different policies aiming at sensible changes in transport behavior and new motor technologies. Each of these scenarios imply different characteristics (a list of the main hypothesis can be found in annex) that are tightly linked to modal shares and demographic dynamics.

In sum, the first scenario is a business as usual (BAU) situation with strict technology standards. This scenario depicts a 48% reduction in emissions whereas the other two scenarios (multimodality and decoupling) represent a reduction of a little over 75%. In the following paragraphs we will first present the details of the BAU scenario and then we will go over the two alternative scenarios.

Promoting strict technology standards (BAU)

The BAU scenario represents a situation where the Speed/GDP elasticity for passengers is of 0.33 and where transport times are stable (1 hour per person per day). This scenario lets us appreciate:  mobility in a situation where there is no major public policy affecting behavior and/or the system's regular performance (continued infrastructure investments and optimization is supposed)



the effects of new motorization technologies on total CO 2 emissions

In this manner the BAU scenario lets us evaluate the contribution of strict and realistic technology standards that -according to our calculations-would lead to half of the reductions of the CO 2 target.

Billion PKm

Interregional Pub. trans.

HST interregional

Plane Interregional road Regional Pub. trans.

Regional road

Urban Pub. trans.

Urban road

H.G. LOPEZ-RUIZ 2010

As we can see in figure 2, if we suppose that hybrid vehicles go into the market in 2010 and electric vehicles are marketed by 2015, the modeled vehicle fleet, for this scenario, would be mainly composed of hybrid vehicles in 2040. This change in technology (coupled to the fact that electricity in France is mainly produced by nuclear reactors) would ensure, in a BAU case almost a 50% reduction in CO 2 emissions. If we take a more detailed look into the details of this scenario, we observe that it is a scenario based on an inelastic market structure largely dependent on private vehicles with high oil prices and a very good offer in public transport. This translates into a scenario that is very dependent on road transport and, thus, dependent on road infrastructures. Therefore, if we suppose that market trends in cars continue to follow current practices, it is more than likely that spatial demand for car use -thus infrastructure needs-will grow accordingly. In order to carry out this assessment, we calculated the investments (see table 1) that would be required throughout the 40 year period, to 2050, for new infrastructures in a BAU scenario (operation costs are not included). In sum, spending on infrastructure would remain at a 1.4% of GDP level (which is roughly the same as in 2007) with most of it going to road infrastructures. Although it would seem like an ideal situation, because we would not be spending more money than we are today, the dilemma behind these results is linked to the fact that it might be a loss of money to have continued high investments on road infrastructure when it can be spent on something else. A BAU scenario with continued investments in road infrastructure would mean a reduction of almost 55 million tonnes of CO 2 (Mt CO 2 ), thus a ratio of 35€ invested in infrastructure per mitigated tCO 2 . Although this ratio is highly speculative and narrow sighted, it is very self illustrative, especially when it will be compared to its value in other scenarios in the following section.

IV. EXPLORING ALTERNATIVE SCENARIOS

Although the BAU scenario results in almost a 50% reduction in GHG emissions, this result is far from the desired 75%. Consequently, the LET-ENERDATA report analyzed two alterative scenarios that look into the effects of public policies aiming at changing behavior. These two scenarios offer a great basis for exploring how sustainable scenarios would shape future investment needs in the transport sector. In the original report, these scenarios are presented as different possibilities to attain important GHG reductions through multimodality and/or decoupling from GDP. The differences between each scenario are linked to the transport structure where: speed/GDP elasticities, modal speeds and transport times differ accordingly to public policy aims. Therefore, each scenario implies different characteristics and thus different types of results that are tightly linked to modal shares and demographic dynamics. Before going into the details of each scenario, the reader can review the mobility results on figure 3.

Billion PKm

Interregional Pub. trans.

HST interregional

Plane Interregional road Regional Pub. trans.

Regional road

Urban Pub. trans.

Urban road H.G. LOPEZ-RUIZ 2010

Promoting green multimodality

In this scenario, market oriented policies constrain the use of high carbon footprint modes which lead to an increase in the use of slower transport options that have smaller carbon footprints. In this scenario, the 75% reduction objective is nearly attained by favoring greener modes through an increase in transport costs according to their speed and associated emissions. As a result, the multimodal scenario shows a change in behavior patterns where the main effects are:



a trade-off between the system's need for speed (coupled to growth),  an increase in transport.

Indeed, since the characteristics of this scenario imply a speed/GDP elasticity equal to zero, translates into an increase in transport times (roughly 1 hour and 20 minutes per person per day). Thus, this scenario is based on market oriented public policies in an infrastructure intensive situation (because transport distances and public transport traffic increase sharply). In this manner, it lets us appreciate that a mix of technology and policy can get us to the wished reduction target but at the cost of slower transports speeds, higher transport times and continued investment needs in infrastructure (see table 2).

Promoting decoupling between transport activities and GDP

The main issue in this scenario is a trade-off between an elevated transport cost and average transport distances. Indeed, transport costs (both in time and in money) are considered to be higher than in the multimodal scenario. This results in economic agents choosing to modify their household/firm locations and develop a proximity intensive way of life. This scenario implies a Speed/GDP elasticity equal to zero but, since transport distances increase less rapidly than in the BAU and the multimodal cases, transport times are reestablished around one hour per person per day. In this manner, the decoupling scenario leads the way to reductions in GHG emissions that go over the 75% objective through market mechanisms, regulation and spatial planning. This new equilibrium based on proximity gives the system a better opportunity for the implementation of low range 0 emission vehicle technologies and also requires lower investments on transport infrastructure.

As we can see in table 2, a decoupling scenario lets us appreciate a situation where mobility increases from the 2000 level (and less than in a BAU or multimodal scenarios) but where infrastructure needs are not as overwhelming as in the two previous scenarios. Nevertheless, we need to take into account that as transport distances get shorter, cities get denser and this implies high costs in urbanism investments (which are not calculated in table 2). In sum, these three scenarios and their assessment gives us a clear view of how the allocation of funds in planning for CO 2 reductions will be a crucial factor. Indeed, the value of CO 2 reductions per euro invested, in a BAU scenario (35€), is a bit higher than that of the multimodality scenario (33€) and higher than the decoupling scenario (20€). In our view, this ratio is important in the sense that the two alternative scenarios present a situation where, even though investments needs are high (maybe even higher than the BAU if we add urban planning costs in the decoupling scenario), the choice of infrastructure in which the community would be investing would seem more sensible. Furthermore, investment calculations for the scenarios clearly illustrate that the implementation of different long-term policy mixes entails a (re-)optimization of agents' (passengers and firms) choices based on transport cost, infrastructure availability and the opportunities offered by transport services. Therefore, these sustainable scenarios imply private social effects linked to welfare variations associated to trade-offs between transport expenditure; house/firm location; accessibility; transport monetary budgets and in fine consumption of goods/services. These variations are important to understand in order to define how acceptability of these different public policies can be increased through adaptive strategies.

V. ADAPTING TO CHANGE

In order to assess how change in behavior will influence adaptive strategies in the long term, it is necessary to take into account how changes in passenger behavior -as a result of public policy-will undoubtedly have an effect on the system. For this, IT-UP can offer some insight into how the (re-)optimization linked to public action in different scenarios can lead to household budget (money and time) reallocation effects that will have an important impact on other sectors (which will have a loopback effect on transport).

As long as the inherent principle of transport policies will be based on rendering high carbon footprint transport less attractive (through cost, speeds, level of service, etc.) than low carbon footprint transport, the results stemming from the policy's macroeconomic changes will most certainly influence microeconomic choices in other domains. These effects will be different for each country, network and set of public policies.

In this manner, if we assess the IT-UP results for the French multimodality scenario, we see that as constraints on speed and emissions come into play as a signal aiming at changing behavior patterns, there is a sharp increase in the use of rail and public transport. This, in turn, implies that average speed in the system should invariably go down and transport times should go up (more or less depending on the evolution of car use elasticity).

This situation seems particularly difficult because it translates into: paying more (for car users) for lower transport speeds (an all modes) and thus loosing potential value added time (VAT) that could be spent increasing revenue. In this setting, we can easily deduct that increasing the sum of goods and services (opportunities) linked to transport activities might help to counterbalance the situation of lost VAT (in other words increasing the numerator in equation 2)

If we follow the same line of reasoning, the logic behind a decoupling scenario is very influenced by proximity services and public policies at play are largely related to spatial planning and infrastructure investment. In a decoupling logic, the main trade-off at play is directly linked to localization strategies and production organization aimed at decoupling transport distances form GDP growth. This entails a densification of main cities and production sites which would, in turn, translate into a sharp increase in the use of urban and regional road networks.

Unlike the multimodality scenario, transport cost characteristics in a decoupling logic lead to more stable transport money budgets because transport distances grow at a slower pace. Nevertheless, if a decoupling scenario is not followed by an adaptive strategy based on a fast increase in the supply of proximity solutions as consumer behavior is modified, an important loss of welfare could be observed.

In sum, a multimodality scenario implies paying more for the same opportunities with higher transport times and, in consequence, a microeconomic equilibrium requires adaptive strategies looking to counterbalance lost welfare by increasing opportunities. In a decoupling scenario, due to the fact that constraints are even higher than in a multimodality scenario, opportunities must increase even more (becoming proximity opportunities) in order to counterbalance overall distance reductions and stay within an equilibrium.

Moreover, the need for adapting to mitigation is reinforced by the fact that as constraints on oil consumption grow, more and more passengers will turn to public transport services. Consequently, a second underlying factor (the first being the before mentioned microeconomic equilibrium hypothesis) that explains the need for planning adaptive strategies is: market power. Indeed, as private vehicle costs rise, public transport use will also rise and, in consequence, this will imply a decrease in price elasticity of demand in public transportation and thus cause a shift in market power. This change in market power will most undoubtedly profit users instead of transport operators. Consequently, the system would be pushed towards a change in how opportunities are conceived by operators and planners. Indeed, as market power shifts, the need for a change in the way that opportunities are assessed, planned and evaluated would become more and more pressing at the risk of welfare loss. As a result, assessment methods would have to start taking into account the time it takes to access opportunities (c.f. the denominator of equation 2).

Currently, certain dense networks are already starting to carry out this type of analysis, for example the Access To Opportunities and Services (ATOS) index (COOPER, S. [START_REF] Cooper | Measuring the accessibility of opportunities and services in dense urban environments: experiences from London[END_REF] proposed by Transport for London in order to improve planning measures. Indeed, just as the UK's planners have begun changing their metrics in order to take into account these new behavior patterns in transport activities, ITS specialist and planners will have to evolve in order to seize the opportunity to offer new services and explore new markets that will be based on a choice model taking into account adaptive strategies that conceive the utility of the representative agent as not only being a function of opportunities but also of the goods and services that are accessible to him in a reasonable lapse of time:

Accordingly, this change in how the utility function of users is integrated into the planning process will imply a differentiation within transport time budgets and how it is perceived by the agent's choice model (c.f. equation 2). In this sense, the choice model would change in order to take into account the effects that access time would have in a situation where we differentiate the time needed to consume and also the time it takes to get to that consumption.

As public policy evolves, changes in the transport system will suppose behavioral modifications. This situation will face planners and industry deciders with a new challenge: to plan according to a utility function that will not only depend on the opportunities an agent has but also on the possibility of actually being able to consume them. In this manner, all changes linked to public policies affecting time use should be accompanied by adaptive strategies.

VI. CLOSING REMARKS

On the basis of the three scenarios, different ways of attaining planned CO 2 reductions were analyzed and discussed. In sum, realistic technological hypothesis show that a 50% reduction in emissions is a clear possibility and that going further based on new technologies would require very big advances in zero emission vehicles.

Nevertheless, in the absence of these new technologies, the remaining reductions in emissions are possible through different types of policy mixes that come down to:



encouraging important modal shifts that would translate into a decrease in total average speed which would in turn make transport times go up,



encouraging modal shift accompanied by a decoupling of transport distances. Consecutively, this would help to maintain stable transport times.

In this setting, this paper offered a brief view of current developments concerning organizational solutions that could lead to a reduction in oil consumption and emissions through important changes in the transport structure and behavior patterns and proposes a quantitative analysis of investment needs for different policy mixes.

On this basis, this paper gives insight on how policies aiming at modifying passenger behavior could heighten the pressure put on infrastructure demand (road, rail, etc depending on the scenario) and will be an important issue in long-term planning for mitigation.

In addition, we would like to identify three main points on the relationship between policy and their impact on adaptive strategies: Mitigation leads to systemic changes. On a regional and interregional level "low-emission high speed transport" for passengers is something that already exists and that will most certainly keep on being promoted as a solution to GHG emissions. Nevertheless, planning for mitigation will most certainly bring about big changes on how public transport is conceived, by planners as well as users, and how it might be better utilized.

Change needs flexibility.

Integrating the user into the planning process will bring about important changes in time use and consumption behaviors accompanied by the continued development of proximity services. All of this implies the need for innovative transport solutions and new consumer services. In this manner, associating freight policy strategy and passenger policy strategy with urban and transport planning will be important in order to gain flexibility whilst continuing to open new markets for new types of behaviors and consumption patterns. These integrated policies for mitigation will undoubtedly require careful planning.

Flexible cities adapt over time.

Although, the actual lack of quick answers to the climate risk problem will most certainly continue to be an important subject in future-studies and even though innovative ideas will help overcome this problem, it will most certainly take time to seriously address all problems involved with transport activities. However, adaptation based planning should not to be underestimated as it offers great opportunities for mitigation acceptability.

Figure

  Figure 1 Passenger mobility BAU

Figure

  Figure 2 Total number of cars per type of motor technology

Figure

  Figure 1 Passenger mobility for all scenarios

  al; LOPEZ-RUIZ & CROZET; SCHADE & HELFRICH et al; SCHIPPER & NG et al; SPERLING

Table 1 Investments in new infrastructure

 1

	Investments Road	1043	21	0,7%	12	0,9%
	Rail	747	15	0,5%	2	0,2%
	Public Trn.	137	3	0,1%	2	0,2%
	Others	-	-	-	1	-
	Total	1927	39	1,4%	18	1,4%
	H.G. LOPEZ-RUIZ 2009
	Note: -

means not applicable -Values are in 2000€ Billions of € Mode 2050 Pegasus Per annum % of GDP % of GDP Year 2007

H.G. LOPEZ-RUIZ 2010

Table 2 Investments in infrastructure for all scenarios

 2

		2050	Per	% of	2050	Per	% of	2050	Per	% of	Year	% of
	Billions of € Mode	Pegasus	annum	GDP	Chronos	annum	GDP	Hestia	annum	GDP	2007	GDP
	Investments Road	1043	21	0,7%	384	8	0,3%	140	3	0,1%	12	0,9%
	Rail	747	15	0,5%	1529	31	1,1%	992	20	0,7%	2	0,2%
	Public Trn.	137	3	0,1%	74	1	0,1%	77	2	0,1%	2	0,2%
	O t h e r s	-	-	-	-	-	-	-	-	-	1	-
	Total	1927	39	1,4%	1987	40	1,4%	1209	24	0,9%	18	1,4%
	H.G. LOPEZ-RUIZ 2009
	Note: -means not applicable -Values are in 2000€


I Christiaans

S B Kenter

H C Brink

T A M Van Os

F Baas

P Van Den Munckhof

A M J Kidd

Dr Theo J M Hulsebos
email: t.j.hulsebos@amc.uva.nl

Germline SMARCB1 mutation and somatic NF2 mutations in familial multiple meningiomas

Background

Multiple meningiomas occur in less than 10% of meningioma patients. Their development may be caused by the presence of a predisposing germline mutation in the neurofibromatosis type 2 (NF2) gene. The predisposing gene in patients with non-NF2associated multiple meningiomas remains to be identified. Recently, SMARCB1 was reported to be a potential predisposing gene for multiple meningiomas in a family with schwannomatosis and multiple meningiomas. However, involvement of this gene in the development of the meningiomas was not demonstrated.

Results

We investigated five affected members of a large family with multiple meningiomas for the presence of mutations in SMARCB1 and NF2. We identified a missense mutation in exon 2 of SMARCB1 as the causative germline mutation predisposing to multiple meningiomas and demonstrated that, in accordance with the two-hit hypothesis for tumourigenesis, the mutant allele was retained and the wild-type allele lost in all four investigated meningiomas. In addition, we identified independent somatically acquired NF2 mutations in two meningiomas of one patient with concomitant losses of the wildtype NF2-allele.

Conclusion

We conclude that, analogous to the genetic events in a subset of schwannomatosisassociated schwannomas, a four-hit mechanism of tumour suppressor gene inactivation, involving SMARCB1 and NF2, might be operative in familial multiple meningiomasassociated meningiomas.

Introduction

Meningiomas are among the most common intracranial primary tumours (20% of all brain tumours), usually occurring as slowly growing sporadic solitary lesions [START_REF] Claus | Epidemiology of intracranial meningioma[END_REF]. They also develop in about 50% of patients with neurofibromatosis type 2 (NF2). The hallmark of NF2 is the presence of bilateral vestibular schwannomas. Schwannomas at other locations are found in more than 50% of patients [START_REF] Evans | A genetic study of type 2 neurofibromatosis in the United Kingdom. I. Prevalence, mutation rate, fitness, and confirmation of maternal transmission effect on severity[END_REF][START_REF] Parry | Neurofibromatosis 2 (NF2): clinical characteristics of 63 affected individuals and clinical evidence for heterogeneity[END_REF][START_REF] Mautner | The neuroimaging and clinical spectrum of neurofibromatosis 2[END_REF]. NF2 patients carry a heterozygous germline mutation in the NF2-gene and the wild-type copy of this gene is often found to be lost in the NF2-associated tumours [START_REF] Bijlsma | Analysis of mutations in the SCH gene in schwannomas[END_REF][START_REF] Mérel | Screening for germ-line mutations in the NF2 gene[END_REF][START_REF] Lamszus | Allelic losses in neurofibromatosis 2-associated meningiomas[END_REF].

Multiple meningiomas occur in less than 10% of patients with meningioma [START_REF] Nahser | Multiple meningiomas. Clinical and computer tomographic observations[END_REF].

These may be NF2-associated and in those cases develop as the consequence of the presence of a predisposing NF2 germline mutation. Non-NF2-associated multiple meningiomas may occur as sporadic or familial cases. The limited number of meningiomas of familial multiple meningioma patients investigated thus far did not display somatic or constitutional NF2 mutations [START_REF] Heinrich | Multiple meningiomas: Investigating the molecular basis of sporadic and familial forms[END_REF][START_REF] Evans | Multiple meningiomas: differential involvement of the NF2 gene in children and adults[END_REF]. The multiple tumours of sporadic patients were found to carry an identical somatic NF2-mutation, which was not present in their constitutional DNAs [START_REF] Heinrich | Multiple meningiomas: Investigating the molecular basis of sporadic and familial forms[END_REF][START_REF] Evans | Multiple meningiomas: differential involvement of the NF2 gene in children and adults[END_REF][START_REF] Stangl | Clonality of multiple meningiomas[END_REF][START_REF] Zhu | Clonal analysis of a case of multiple meningiomas using multiple molecular genetic approaches: pathology case report[END_REF][START_REF] Eckstein | Multiple meningiomas in brain and lung due to acquired mutation of the NF2 gene[END_REF]. This might be explained by spread via the cerebrospinal fluid or distant metastasis (ref 13) of a single tumor or as a consequence of mosaicism with undetectable contribution of the NF2-mutation to the constitutional DNA. Alternatively, the non-NF2 sporadic and familial cases collectively suggest that a gene different from NF2 is implicated in the development of non-NF2-associated multiple meningiomas. This contention is supported by an earlier linkage study in a family with meningiomas in which a 15 cM region around and including NF2 was excluded from harbouring the meningioma predisposing gene [START_REF] Pulst | Familial meningioma is not allelic to neurofibromatosis 2[END_REF].

SMARCB1 is a predisposing gene in schwannomatosis, which is characterized by the presence of multiple schwannomas in the absence of vestibular schwannomas. The gene is involved in about 30-40% of the familial cases, but in no more than 10% of the sporadic cases. In the schwannomas of these patients, the wild-type copy of SMARCB1 is found to be inactivated, usually by deletion. In addition, somatically acquired NF2mutations have been found in schwannomas of patients with a germline SMARCB1mutation [START_REF] Hulsebos | Germline mutation of INI1/SMARCB1 in familial schwannomatosis[END_REF][START_REF] Boyd | Alterations in the SMARCB1 (INI1) tumor suppressor gene in familial schwannomatosis[END_REF][START_REF] Hadfield | Molecular characterisation of SMARCB1 and NF2 in familial and sporadic schwannomatosis[END_REF][START_REF] Sestini | Evidence of a four-hit mechanism involving SMARCB1 and NF2 in schwannomatosis-associated schwannomas[END_REF].

We wondered whether SMARCB1 might be a predisposing gene in multiple meningiomas as well. Support for this candidacy is given by the location of SMARCB1, which is proximal to marker D22S1, i.e. outside the region excluded by linkage analysis [START_REF] Pulst | Familial meningioma is not allelic to neurofibromatosis 2[END_REF]. Furthermore, somatic SMARCB1 mutations have been shown to occur in sporadic meningiomas, albeit at low frequency, i.e. less than 3% of tumours [START_REF] Bruder | The mouse ortholog of the human SMARCB1 gene encodes two splice forms[END_REF][START_REF] Schmitz | INI1 mutations in meningiomas at a potential hotspot in exon 9[END_REF][START_REF] Rieske | Molecular heterogeneity of meningioma with INI1 mutation[END_REF]. Evidence for the involvement of SMARCB1 in multiple meningiomas was recently reported by Bacci et al. [START_REF] Bacci | Schwannomatosis associated with multiple meningiomas due to a familial SMARCB1 mutation[END_REF], who showed the inheritance of a germline SMARCB1 mutation in a family with schwannomatosis and multiple meningiomas. However, the mutational status of SMARCB1 (and of NF2) in the meningiomas of the patients was not clarified. In another recent study, no germline SMARCB1 mutation could be detected in 47 patients with multiple meningiomas, including 8 familial cases [START_REF] Hadfield | SMARCB1 mutations are not a common cause of multiple meningiomas[END_REF].

In this study, we investigated a large family with three members affected by multiple meningiomas and two with a single meningioma. We identified the causative germline SMARCB1 mutation segregating with (multiple) meningiomas in this family and demonstrated inactivation by deletion of the wild-type copy of SMARCB1 and additional bi-allelic and independent inactivation of NF2 in meningiomas of the patients.

Materials and Methods

Patients

Informed consent was obtained from all family members for using their body materials.

The proband (III-6 in Figure 1) was referred to our outpatient clinic with a suspected diagnosis of neurofibromatosis type 2 because of multiple meningiomas. At the age of 34 she presented with epilepsy and computer tomography (CT) of the brain showed three extra-axial lesions along the falx cerebri, suspected for meningiomas. Two lesions were resected and pathological examination confirmed the diagnosis meningioma.

At the age of 51 years, our proband developed sensory problems and paraplegia of the legs. Magnetic resonance imaging (MRI) of the brain and spinal cord showed an intradural extramedullary lesion at Thoracic level (T) 7, suspected for meningioma. The lesion was removed and turned out to be a psammomatous meningioma, World Health Organization (WHO) grade I. Her family history revealed a twin sister (III-7 in Figure 1) with a chest wall schwannoma at age 37. At age 49, she developed epilepsy and brain MRI showed multiple extra-axial lesions, which were on histology transitional/mixed meningiomas grade I. She underwent additional surgery for a spinal fibroblastic meningioma at T4, multiple schwannomas of the chest wall and elbow, and was diagnosed with multiple spinal lesions suspected for schwannomas. The father of these twin sisters died at age 63 from coronary artery disease. Two of his ten siblings had been operated for meningioma. An uncle of our proband (II-6 in Figure 1) developed left-sided deafness caused by a cerebellopontine angle mass, suspected for vestibular schwannoma, at age 56. At age 64, a spinal fibroblastic meningioma at T1 was removed and, more recently, at age 77, he developed gait difficulties and MRI showed multiple spinal intradural extramedullary lesions, suspected for meningiomas. An aunt of our proband (II-9 in Figure 1) was operated on a spinal psammomatous meningioma at T10 at age 53.

A fibrous meningioma at T6 was removed from the spinal cord of her daughter (III-1 in Figure 1) at the age of 40 years.

None of the affected family members had neurofibromas, café-au-lait spots or freckling. Their clinical characteristics are summarized in Table 1.

DNA and RNA samples

A frozen tissue sample of a brain meningioma M1 of patient III-6 and FFPE tissue samples of one meningioma each of patients II-6, II-9, and III-1 and the brain and spinal cord meningiomas (M1, M2) of patient III-6 were available for molecular analysis. DNA was extracted and purified from blood samples and the tumour tissue samples according to standard methods. DNA extracted from the meningioma of patient II-9 was too heavily degraded for reliable molecular analyses.

Total RNA was extracted and purified from another part of the frozen tissue sample of meningioma M1 of patient III-6 using TRIzol® LS Reagent according to the protocol of the manufacturer (Invitrogen, Leek, The Netherlands). One µg of total RNA was used to synthesize cDNA with random priming according to standard methods.

Microsatellite analysis

Genotyping of family members and loss of heterozygosity (LOH) analysis of tumours were performed by using the chromosome 22-specific microsatellite markers of the ABI Prism® Linkage Mapping Set version 2.5-HD5 and additional marker D22S929. Primer sequences for the latter marker were taken from Legoix et al. [START_REF] Legoix | Characterisation of 16 polymorphic markers in the NF2 gene: application to hemizygosity detection[END_REF]. Data were analyzed with Genemapper software version 4.0. (Applied Biosystems, Foster City, CA, USA).

Mutation analysis

SMARCB1 and NF2 were sequenced by using genomic or cDNA as substrate for amplification by PCR. Primer sequences for mutation analysis of the SMARCB1 and NF2 exons have been published previously [START_REF] Hulsebos | Germline mutation of INI1/SMARCB1 in familial schwannomatosis[END_REF]. For improved performance, some SMARCB1 primers were newly designed (sequences available on request). In degraded DNA extracted from FFPE material, the SMARCB1 exon 2 segment harbouring the missense mutation was sequenced by using forward primer 5'-CCCTTATAATGAGCCTTCTTGCT-3' and reverse primer 5'-TCTTCCACAGTGGCTAGTCG-3', generating a PCR-product of 116 bp.

The cDNA product from meningioma M1 of patient III-6 was sequenced by using forward primer 5'-GACGGCGAGTTCTACAT-3' in exon 1 and reverse primer 5'-TTTTACCATGTGACGATGCAA-3' at the 3'-end of exon 2, generating a PCR-product op 148 bp. All sequence reactions were performed using ABI Big Dye v3.1 chemistry, and the products were sequenced with an ABI 3730 capillary system (Applied Biosystems, Foster City, CA, USA). Sequences were analyzed with CodonCode Aligner (CodonCode Corporation, Dedham, MA, USA).

The HomoloGene program in PubMed was used to perform amino acid sequence alignments (http://www.ncbi.nlm.nih.gov/pubmed). The PolyPhen and SIFT programs were used to predict the impact of amino acid substitutions on protein function and 10 structure (http://genetics.bwh.harvard.edu/pph and http://sift.jcvi.org, respectively).

Results

Sequencing of the nine exons of SMARCB1 in constitutional DNA of proband III-6 revealed a missense mutation in exon 2, c.143C>T, P48L (Figure 2), which also proved to be present in constitutional DNA of the other affected family members (III-7, II-6, II-9, and III-1). The mutation was not found in constitutional DNA of more than 100 control individuals. The proline residue that is replaced by leucine in the resulting SMARCB1 protein is evolutionary conserved across all species for which sequence information is available (data not shown). In silico analyses with the Polyphen and SIFT programs predicted that the amino acid substitution affects SMARCB1 protein function and is probably damaging.

The four meningiomas that were available for molecular analysis all showed loss of the wild-type C allele, as demonstrated in Figure 2 for the brain (M1) and spinal cord (M2) meningioma of the proband (III-6). In accordance with this, genotyping with flanking markers revealed retention of the chromosome 22 segment containing the mutant T-allele in the investigated tumours, as exemplified in Figure 2 for marker D22S1174. The data are summarized in Figure 1.

To investigate whether the retained SMARCB1-allele was transcribed, we extracted total RNA from frozen tissue of meningioma M1 and reverse-transcribed it into cDNA. The cDNA sequence demonstrates that a stable mRNA is transcribed from the retained mutant allele (Figure 2).

Schwannomatosis-associated schwannomas harbour somatically acquired NF2

mutations. Considering the apparent analogy with schwannomatosis, we wondered whether these mutations might also be present in the meningiomas of the family under study. Although attempts were made to sequence NF2 in all available meningiomas, reliable sequences could only be derived from the frozen tumour M1 and the freshlyfixed tumour M2, both of proband III-6. We found a missense mutation in exon 4 of meningioma M1 (c.422T>G, p.L141R) and a nonsense mutation in exon 8 (c.773G>A, p.W258X) of meningioma M2, both being absent in the corresponding constitutional DNA of the patient (Figure 3). The leucine residue at position 141 is evolutionary conserved (data not shown). The Polyphen and SIFT programs predicted that the amino acid substitution caused by the missense mutation affects NF2 protein function and is probably damaging. The sequence recordings indicate loss of the wild-type allele in both tumours. This is substantiated by LOH analysis with the intragenic NF2 marker D22S929, which shows considerable loss of the 132 bp allele in tumour M1 as well as tumour M2 (Figure 3). Finally, we sequenced all exons of the NF2 gene in constitutional DNA of the affected family members (III-6, III-7, II-6, II-9, III-1), but did not detect any deviation from the wild-type sequence. In addition, we performed multiple ligationdependent probe amplification (MLPA) analysis of the NF2 gene in constitutional DNA of patient III-7. However, this analysis did not reveal any deletions or duplications.

Discussion

We identified the germline mutation c.143C>T in SMARCB1 that most probably predisposes to the development of multiple meningiomas. The mutation segregates with the presence of (multiple) meningiomas in this family and, in accordance with Knudsons' two-hit hypothesis for tumourigenesis [START_REF] Knudson | Mutation and cancer: statistical study of retinoblastoma[END_REF], remains in the four investigated meningiomas, each with loss of the wild-type SMARCB1 allele by LOH (Figure 2). The causative nature of this missense mutation may be inferred from our in silico analyses, which demonstrated that the proline residue at position 48 is evolutionary conserved and that the change to leucine affects SMARCB1 protein function and is probably damaging.

The importance of proline at that position is furthermore supported by the finding of another missense mutation causing its replacement (c.142C>T, p.P48S) in a malignant rhabdoid tumour of the kidney [START_REF] Sévenet | Spectrum of hSNF5/INI1 somatic mutations in human cancer and genotype-phenotype correlations[END_REF]. Another missense mutation in the SMARCB1 gene, c.92A>T, p.E31V, was recently identified as the predisposing genetic event in a family with schwannomatosis and multiple meningioma [START_REF] Bacci | Schwannomatosis associated with multiple meningiomas due to a familial SMARCB1 mutation[END_REF]. RNA studies in whole blood of two patients in this family demonstrated absence of transcripts from the mutant SMARCB1-allele. We, however, detected a stable transcript derived from the mutant SMARCB1-allele that retained in meningioma M1 of patient III-6 (Figure 2). Accordingly, we found uniform staining of nuclei with a SMARCB1 antibody (anti-BAF47) in all four meningiomas, suggesting that an intact SMARCB1 protein was formed (data not shown).

The presence of a left sided cerebellopontine angle mass, probably a vestibular schwannoma, in patient II-6 and of multiple schwannomas in patient III-7 suggest that the SMARCB1 mutation in this family may predispose to the development of schwannomas as well. However, the vestibular schwannoma was diagnosed at age 56 and such a sporadic unilateral tumour is not uncommon at that age in the general population [START_REF] Baser | Increasing the specificity of diagnostic criteria for schwannomatosis[END_REF].

Unfortunately, the schwannomas were not available to further investigate the possible involvement of SMARCB1. A schwannoma analyzed in the family with schwannomatosis and multiple meningiomas indeed displayed bi-allelic inactivation of SMARCB1 [START_REF] Bacci | Schwannomatosis associated with multiple meningiomas due to a familial SMARCB1 mutation[END_REF].

Two other schwannomatosis families, each with one patient developing one or two meningiomas, have been reported. However, the status of SMARCB1 in these tumours was not investigated [START_REF] Boyd | Alterations in the SMARCB1 (INI1) tumor suppressor gene in familial schwannomatosis[END_REF][START_REF] Hadfield | Molecular characterisation of SMARCB1 and NF2 in familial and sporadic schwannomatosis[END_REF][START_REF] Evans | Spinal and cutaneous schwannomatosis is a variant form of type 2 neurofibromatosis: a clinical and molecular study[END_REF][START_REF] Maccollin | Familial schwannomatosis: exclusion of the NF2 locus as the germline event[END_REF]. Considering the frequent occurrence of meningiomas in the general population (1), it cannot be excluded that their association with schwannomatosis in the latter families was coincidental. On the other hand, the missense mutation in SMARCB1 in our family, affecting amino acid residue 48, and most probably also in the family presented by Bacci et al. [START_REF] Bacci | Schwannomatosis associated with multiple meningiomas due to a familial SMARCB1 mutation[END_REF], affecting nearby amino acid residue 31, clearly predispose to the development of multiple meningiomas. Therefore, it is possible that the preference for meningioma development is, at least in part, determined by type and location of the SMARCB1 mutation. Alternatively, the cooccurrence of schwannomas and meningiomas in some SMARCB1 families and the sole occurrence of schwannomas in most other SMARCB1 families may point to the existence of modifying genes segregating in these families and affecting the phenotypic expression of the SMARCB1 mutation. Our finding and that of Bacci et al. [START_REF] Bacci | Schwannomatosis associated with multiple meningiomas due to a familial SMARCB1 mutation[END_REF] that meningiomas encompass the schwannomatosis tumour spectrum may require reconsideration of the diagnostic criteria for this disease. Further investigation of the possible association of type and location of the SMARCB1 mutation and the development of multiple meningiomas and of possible modifying factors will require the identification and clinical evaluation of additional carriers of the mutated gene in our family and in other multiple meningiomas patients and families.

We identified additional independent somatic mutations in the NF2 gene in the brain (M1) and spinal cord (M2) meningiomas of patient III-6 (Figure 3). The missense mutation in exon 4 in meningioma M1 caused replacement of the evolutionary conserved leucine residue at position 141 in the NF2 protein and its change into arginine affects, according to the in silico analyses, protein function and is probably damaging. The importance of leucine at position 141 is furthermore supported by the finding of another missense mutation causing its replacement (c.422T>C, p.L141P), which was reported as a constitutional NF2 mutation in a patient with bilateral schwannomas [START_REF] Ahronowitz | Mutational spectrum of the NF2 gene: a meta-analysis of 12 years of research and diagnostic laboratory findings[END_REF]. The exon 8 mutation in meningioma M2 is a typical NF2 nonsense mutation, causing truncation of the protein [START_REF] Ahronowitz | Mutational spectrum of the NF2 gene: a meta-analysis of 12 years of research and diagnostic laboratory findings[END_REF]. In accordance with Knudsons' two-hit model, loss of the wild-type copy of NF2 by LOH occurred in both tumours [START_REF] Knudson | Mutation and cancer: statistical study of retinoblastoma[END_REF]. The finding of two causative hits (NF2 mutation and deletion) in these meningiomas and our failure to detect a constitutional NF2 mutation or deletion in the affected family members strongly argue against this gene as being the multiple meningioma predisposing gene in this family.

Summarizing our data, we conclude that, analogous to the genetic events in a subset of schwannomatosis-associated schwannomas [START_REF] Boyd | Alterations in the SMARCB1 (INI1) tumor suppressor gene in familial schwannomatosis[END_REF][START_REF] Hadfield | Molecular characterisation of SMARCB1 and NF2 in familial and sporadic schwannomatosis[END_REF][START_REF] Sestini | Evidence of a four-hit mechanism involving SMARCB1 and NF2 in schwannomatosis-associated schwannomas[END_REF], a four-hit mechanism of tumour suppressor gene inactivation, involving SMARCB1 and NF2, might also be operative in familial multiple meningiomas-associated meningiomas. In a recent study, no germline SMARCB1 mutations were detected in 47 patients with multiple meningiomas, including eight familial cases [START_REF] Hadfield | SMARCB1 mutations are not a common cause of multiple meningiomas[END_REF]. Germline SMARCB1 mutations are infrequently found in sporadic schwannomatosis patients (less than 10% of cases). It is possible that, as in familial schwannomatosis (30-40% of cases), SMARCB1 involvement is more frequent in familial multiple meningiomas. Alternatively, other genes might be implicated as predisposing genetic factors in both diseases.

Licence for Publication

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in JMG and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence (http://group.bmj.com/products/journals/instructions-for-authors/licenceforms).

Legends to the figures

Figure 1

 1 Figure 1

Figure 2

 2 Figure 2

Figure 3

 3 Figure 3

Table 1 :

 1 Clinical characteristics of affected family members

	Patient	Sex Age (y)	Presenting symptom	Meningiomas	Schwannomas
		(M/F) at diagnosis		(n, location)	(n, location)
	III-6	F	34	Epilepsy	3 cranial, 1 spinal	-
	III-7	F	37	Chest wall mass	Multiple cranial,	Multiple chest wall,
					1 spinal	multiple spinal,
						1 elbow
	II-6	M	56	Deafness	Multiple spinal	1 vestibular
	II-9	F	53	Paresis legs	1 spinal	-
	III-1	F	40	Sensory problems legs	1 spinal	-

Acknowledgements:

We thank the family members for their participation in this study. We are indebted to Dr. D. Troost (Neuropathology Department, Academic Medical Center, Amsterdam), Dr M.G Havenith (Pathology Department, Isala Clinics, Zwolle), D. Stomphorst (Pathology Department, Leiden University Medical Center, Leiden), and M. Waaijer (Pathology Department, Medical Center Alkmaar, Alkmaar) for providing tumour materials.

Competing interests: None to declare

Patient consent: Obtained


Sara Molatore

Ilaria Marinoni

Misu Lee

Elke Pulz

Ettore Maria Rosaria Ambrosio

Degli Uberti

Maria Chiara Zatelli

Dr Natalia S Pellegata
email: natalia.pellegata@helmholtz-muenchen.de

Ettore C Degli Uberti

A novel germline

Keywords: Multiple endocrine neoplasia type 4, p27, mutation screening, functional characterization Multiple endocrine neoplasia type 4, p27, mutation screening, functional characterization

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

We recently demonstrated that germline mutation in Cdkn1b (OMIM: *600778), which encodes the p27 cyclindependent kinase (Cdk) inhibitor, causes a multiple endocrine neoplasia (MEN)-like syndrome in the rat (Pellegata et al., 2006). Following this discovery, we identified a germline heterozygous nonsense mutation at codon 76 (c.692G>A, p.W76X) of the human CDKN1B gene in several members of a family with a variant of the MEN1 syndrome, thereby demonstrating that CDKN1B is a tumor susceptibility gene also in humans (Pellegata et al., 2006). Subsequently, an independent study identified a germline CDKN1B frameshift mutation at codon 25 (c.59_77dup, c.K25fs) in a female patient with small-cell neuroendocrine cervical carcinoma, Cushing's disease, and hyperparathyroidism [START_REF] Georgitsi | Germline CDKN1B/p27Kip1 mutation in multiple endocrine neoplasia[END_REF]. This mutation was associated with loss-of-heterozygosity (LOH) of the wild-type CDKN1B allele in the patient's cervical carcinoma. Recently, three potentially pathogenic changes in CDKN1B (c.-7G>C; c.283C>T, p.P95S; c.595T>C, p.X199QextX*60) were identified in patients with hereditary predisposition to MEN1-like tumors or primary hyperparathyroidism (1ºHPT) [START_REF] Agarwal | Rare germline mutations in cyclin-dependent kinase inhibitor genes in multiple endocrine neoplasia type 1 and related states[END_REF]. In vitro, both c.-7G>C and X199Q variants were expressed at reduced level compared to wild-type p27 (p27wt), while p27P95S showed decreased binding affinity for Grb2, an adaptor protein involved in the activation of Ras signalling [START_REF] Moeller | p27Kip1 inhibition of GRB2-SOS formation can regulate Ras activation[END_REF]. Therefore, CDKN1B mutations, although rare, do occur in patients with MEN1related states, the so called MEN4 syndrome (OMIM: #610755). Due to the small number of mutation-bearing patients so far reported, the phenotypic features associated to MEN4 are still undefined. Moreover, the limited molecular characterisation of the naturally-occurring CDKN1B mutations identified hinders a thorough understanding of the role of p27 in neuroendocrine tumors predisposition.

The aim of our study was to search for new CDKN1B mutations and, if found, to functionally characterize them in vitro. We report here a novel CDKN1B mutation found in a patient with multiple endocrine tumors. Functional analysis of two mutated p27 proteins (the novel we identified and one we previously reported) regarding subcellular localization, protein binding and degradation kinetic, growth suppression and apoptosis are presented.

METHODS

Patients and DNA analysis

The study was approved by the Ethics Committee of the University of Ferrara, Italy and informed written consent was obtained from all patients. Twenty-seven Italian patients displaying a MEN1-like phenotype (hyperparathyroidism, neuroendocrine tumors, pituitary adenoma), but lacking a MEN1 gene mutation, were screened for mutations in CDKN1B (GenBank entry NM_004064.3) as previously reported (Pellegata et al., 2006). To determine the frequency of the c.678C>T allelic variant found in an Italian patient we obtained and analyzed unrelated control DNA samples from the Institute of Human Genetics, Helmholtz Zentrum München, which comprise 370 healthy Germans (KORA Study Group) [START_REF] Filipiak | Farming, rural lifestyle and atopy in adults from southern Germany--results from the MONICA/KORA study Augsburg[END_REF]. Loss of heterozygosity (LOH) analysis was performed as previously reported (Shyla et al., 2010) using the chromosome 12p markers D12S391, D12S358, and D12S1580.

Plasmid constructs and antibodies

The two described mutations were introduced by site-directed mutagenesis (Quikchange II Site-Directed Mutagenesis Kit, Stratagene) in the wild-type human CDKN1B cDNA cloned in a pEYFP backbone, kindly provided by J. Slingerland (University of Miami, FL). Primary antibodies used were: anti-p27 (clone 57) and anti-GFP tag (BD Biosciences); anti-Skp2 (H-345); anti-Cdk2 (D-12); anti-Cdk4 (C-22); anti-Cyclin D1 (H-295); anti-Cyclin E (M-20); α-tubulin (all from Santa Cruz Biotechnology).

Cell culture, transfections, drug treatments

HeLa and MCF7 cell lines were respectively maintained in DMEM or RPMI 1640 media supplemented with 10% fetal bovine serum, 20 mM L-glutamine, 100 units/ml of penicillin G sodium, and 100 µg/ml streptomycin (all Invitrogen). The GH3 cell line was maintained in F-12K medium supplemented with 15% horse serum, 2.5% fetal bovine serum, 20 mM L-glutamine, 100 units/ml of penicillin G sodium, and 100 µg/ml streptomycin. For p27 half-life determination in exponentially growing cells, cycloheximide (CHX, Sigma-Aldrich, 25 µg/ml) was added to the cells for the indicated times. Transfections were carried out as already reported (Pellegata et al., 2006). When establishing stable transfectants, GH3 cells were selected by adding 0.4 mg/ml G418 to the culture medium for 2-3 weeks and individual colonies picked and expanded in presence of G418.

RNA interference

For RNAi studies, short interfering RNA (siRNA) duplexes specific for SKP2 (n=4; siGenome SMARTpool, Dharmacon) and KPC1 (n=2; ID 133486, 133487 Ambion) and scrambled siRNA (Ambion) were obtained and transfected into MCF7 cells using X-tremeGENE reagent (Roche) together with 0.03 µg of plasmid DNA.

Protein extraction, immunoblotting and immunoprecipitations

Protein extraction and immunoblotting were carried out as already reported (Pellegata et al. 2006). For IP, 500 µg of total protein were incubated with 5 µl of anti-YFP tag antibody (Clontech-Takara Bio Europe) and 20 µl of agarose resin (Santa Cruz) overnight at 4 °C. For GST-pull down assay, 500 µg of total protein were incubated overnight at 4° with 2 µg of recombinant Cdk2-GST (Abnova) and 20 µl of Glutathione Agarose (Santa Cruz).

Intracellular localization and apoptosis assay

p27 knock-out (-/-) MEFs were obtained from 13 days old embryos upon trypsin digestion for 15 min at 37ºC. Cells were grown in DMEM supplemented with 10% fetal bovine serum, 20 mM L-glutamine, 100 units/ml of penicillin G sodium, and 100 µg/ml streptomycin (all Invitrogen). For localization experiments, MEFs were grown on coverslips, transfected and processed for immunocytochemistry as described (Pellegata et al., 2006) with minor modifications. For the apoptosis assay, HeLa cells were transfected while exponentially growing on coverslips. At 24h or 48h post-transfection cells were fixed and nuclei were stained with DAPI.

Clonogenic assay and growth curve

To examine clonogenic activity, GH3 cells were plated (500,000 per well) in 6-well plates and transfected with p27wt, p27W76X or p27P69L using Fugene-6 (Roche). The day after the transfection, the cells were diluted 1:6 and, starting from the day after, selected for 6 weeks by adding 0.4 mg/ml G418 to the culture medium and stained with 0.3% crystal violet in 30% ethanol. Colonies that contained 50 cells were scored. p27W76X GH3 stable clones, and untrasfected GH3 cells were plated in duplicate (100,000 per well) in 12-well plate, in full medium plus 400 µg/ml G418. The number of cell was counted every two days over a 9 day-period using a cell counter.

Immunohistochemistry

The two described mutations were introduced by site-directed mutagenesis in the wild-type human CDKN1B cDNA cloned into the pEYFP (YFP, yellow fluorescent protein) backbone. Immunohistochemistry were performed using a monoclonal anti-p27 antibody (BD Biosciences) as already reported (Pellegata et al. 2006).

.

RESULTS

Germline genetic changes and clinical presentation

We screened the genomic DNA of 27 patients for CDKN1B germline changes and found a previously unreported variant at codon 69 (c.678C>T, p.P69L) in patient FL, a 79-year-old Caucasian female (Fig. 1A). This change was not reported in the single nucleotide polymorphism (SNP) database and was not observed in 370 unrelated healthy Caucasian controls, confirming that it is neither a polymorphism nor a rare variant. This patient presented with bilateral multiple lung metastases of bronchial carcinoid and type 2 diabetes mellitus. A sellar magnetic resonance imaging (MRI) revealed a pituitary microadenoma. Clinical work-up did not display any alteration in pituitary hormone levels, indicating the presence of a non functioning pituitary microadenoma. This patient had a history of endocrine and non-endocrine malignancies: at age 67 she was admitted for surgical resection of multiple typical bronchial carcinoids and a subcutaneous epigastric lipoma, and she was subjected to left parathyroidectomy because of a parathyroid adenoma causing 1ºHPT. At age 64 a total thyroidectomy had been performed due to a papillary thyroid carcinoma with neck lymphnode metastases (pT1bN1M0). The patient had no family history of endocrine tumors. Her relatives refused to undergo genetic testing and their phenotype could not be determined.

Functional characterization

In our previous work we identified a heterozygous nonsense mutation at codon 76 (W76X) in a proband with GH-secreting pituitary adenoma and 1ºHPT, and in her sister displaying renal angiomyolipoma (Pellegata et al., 2006). Upon transfection into normal rat fibroblasts, a Myc-tagged p27W76X protein localized to the cytoplasm (Pellegata et al., 2006). Although the renal tumor showed no p27 expression we have now detected p27W76X expression in the normal kidney of the mutation-positive woman, where it localizes to the cytoplasm (see Fig. 4A-B).

To investigate which functional properties of p27wt are affected by P69L and W76X changes, we generated plasmid vectors that expressed both p27 mutants as fusion proteins with the YFP tag located at the N-terminus [START_REF] Connor | CRM1/Ran-mediated nuclear export of p27(Kip1) involves a nuclear export signal and links p27 export and proteolysis[END_REF]. Transfection of these constructs in MCF7, HeLa and the p27-negative GH3 pituitary adenoma cells confirmed the expression of the fusion proteins in vitro and showed that p27P69L is consistently expressed at reduced level compared to p27wt in all cell lines (Fig. 1B). Following transfection of exponentially growing p27-/-mouse embryonal fibroblasts (to avoid any possible interaction with endogenous p27), p27P69L localized mainly to the nucleus, like p27wt (Fig. 1C), although there was a higher proportion of cytoplasmic localization (average of YFP-positive cells showing cytoplasmic localization of the transfected p27P69L protein: 22.6%) compared to p27wt (average of YFP-positive cells showing cytoplasmic localization of the transfected p27wt protein: 8.2%). As expected, p27W76X was cytoplasmic (Fig. 1C).

p27P69L is consistently expressed at lower steady-state level compared to p27wt. This could be due to enhanced protein degradation, the mechanism responsible for p27 down-regulation in many human tumors [START_REF] Pagano | Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27[END_REF][START_REF] Loda | Increased proteasome-dependent degradation of the cyclin-dependent kinase inhibitor p27 in aggressive colorectal carcinomas[END_REF]. Exponentially growing MCF7 cells transfected with p27wt, p27P69L or p27W76X were incubated with Cycloheximide (CHX) to block new protein synthesis and analyzed at different time points thereafter. Up to 8h post-treatment we observed no change in p27wt and p27W76X amount, while p27P69L showed reduced expression after 6h (Fig. 2A). This indicates that p27P69L is degraded slightly faster than p27wt.

siRNA-mediated knock-down of the ubiquitin ligases that mediate p27wt degradation (i.e. SKP2, which acts in the nucleus in G1-S, and KPC1, which works in early G1 in the cytoplasm) [START_REF] Carrano | SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27[END_REF][START_REF] Kamura | Cytoplasmic ubiquitin ligase KPC regulates proteolysis of p27(Kip1) at G1 phase[END_REF] showed that either both these degradation pathways, or just KPC1, play a role also in the degradation of p27P69L and p27W76X, respectively (since p27W76X is cytoplasmic, we only tested the effect of KPC1 knockdown) (Fig. 2B). Co-transfection of the p27 proteins with scrambled siRNA confirmed the specificity of the siSKP2 and siKPC1 molecules (Fig. 2C).

Figure 2

Degradation kinetics of the p27W76X and p27P69L mutant proteins and pathways of degradation. A, p27P69L is slightly more unstable than p27wt. Asynchronously growing MCF7 cells were transiently transfected with the different constructs and 24 h later treated with CHX for the indicated times. Western blotting (WB) was performed using an anti-p27 antibody. The transfected (Tx) exogenous p27 proteins and the endogenous (Endo) p27 are indicated on the left. To control for equal loading the membranes were probed with anti-α-tubulin antibody. B, Degradation of p27W76X and p27P69L is in part mediated by Skp2 and KPC1. MCF7 cells were transfected with siRNA against Skp2 or KPC1 together with expression plasmids for YFP-p27wt, -p27W76X and -p27P69L and analyzed 48 h later by western blotting for p27. Transfected (Tx) exogenous p27 proteins and the endogenous (Endo) p27 are indicated on the left. C, Scrambled siRNA (Scr. siRNA) has no effect on the expression of p27 proteins. Only the transfected proteins are shown. Considering the crystal structure of the p27-CyclinA-Cdk2 protein complex, the P69L change affects one of the six amino acids involved in the direct binding to Cdk2 [START_REF] Russo | Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex[END_REF]. To verify whether this mutation indeed alters the binding of p27 to Cdk2, we performed Cdk2 pull-down assays using HeLa cells transiently-transfected with p27P69L or control p27wt. Associated p27 proteins were detected by immunoblotting. While p27wt binds Cdk2, p27P69L does not bind to the kinase (Fig. 1D).

p27W76X retains most of the cyclin/Cdk binding domain of p27 (aminoacids 28 to 90), therefore it could bind to and sequester in the cytoplasm the canonical p27 partners: Cyclins E and D1, Cdk2 and Cdk4. Upon transfection of proliferating HeLa cells, we immunoprecipitated (IP) p27W76X and detected interacting Cyclins and Cdks by immunoblotting with specific antibodies. A minimal fraction of p27W76X binds to Cyclin D1, while there is no association with the other molecules (Fig. 3A). Similar results were also obtained using GH3-derived clones stably and constitutively expressing p27W76X (see below), demonstrating that the lack of association with partner proteins is not due to transient p27W76X expression nor to interference by endogenous p27wt.

p27 is a negative regulator of cell cycle progression, and its loss is associated with disease progression and unfavourable outcome in many cancer types [START_REF] Chu | The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy[END_REF]. To determine the effect of P69L and W76X mutations on cell growth, we performed a clonogenic assay using p27-negative GH3 cells. The results showed that p27wt inhibits the growth of these cells while p27W76X does not. p27P69L was less efficient than p27wt at suppressing cell growth (Fig. 3B).

Figure 3

Effect of the p27 mutations on protein binding, cell growth and apoptosis. A, Asynchronously growing HeLa cells were transfected with p27W76X and collected 24 h later. Five hundred microgr of the lysate were immunoprecipitated (IP) o.n. with the anti-YFP antibody. Fifty microgr of the original lysate (Input) and the entire IP (Ip YFP) were analyzed by Western blotting (WB) with antibodies against the indicated proteins. B, p27-negative GH3 cells were transfected with the indicated expression plasmids and then selected in G418-containing media for 3 weeks. To verify that the long-term expression of p27W76X has no effect on cell proliferation, we picked and expanded several stably-transfected, GH3-derived clones. Selected representative clones were employed for growth curves over a period of 9 days. In agreement with the clonogenic assay results, constitutive overexpression of p27W76X did not affect GH3 cell proliferation (Fig. 3C).

Tumor suppression by p27wt works through inhibition of cell proliferation, but also induction of apoptosis in some cells types [START_REF] Katayose | Promoting apoptosis: a novel activity associated with the cyclin-dependent kinase inhibitor p27[END_REF]. We assessed whether W76X and P69L mutations interfere with the proapoptotic activity of p27wt using HeLa cells as experimental model, as these cells undergo apoptosis upon p27wt overexpression [START_REF] Nickeleit | Argyrin a reveals a critical role for the tumor suppressor protein p27(kip1) in mediating antitumor activities in response to proteasome inhibition[END_REF]. DAPI staining showed that p27P69L expression induced apoptosis at both 24h and 48h post-transfection, similarly to p27wt, whereas p27W76X lost this ability (Fig. 3D).

p27 expression in patients tissues

We analyzed the expression of p27 in the parathyroid adenoma and one bronchial carcinoid of the patient carrying the p27P69L mutation using IHC. Parathyroid adenoma cells showed weak p27 nuclear staining in <1% of the tumor cells (interspersed normal endothelial cells served as controls) (Fig. 4C-D). The parathyroid adenoma of a mutation-negative control individual exhibited strong p27 nuclear positivity in virtually all cells. A bronchial carcinoid of the P69L mutation-positive patient showed virtually no p27 staining when compared to a bronchial carcinoid of a mutation-negative individual (Fig. 4E-F). To determine the cause of the reduced p27 expression level in the tumors of this patient, we extracted DNA from tumor and adjacent normal cells and performed LOH analysis by amplifying three microsatellite markers flanking the p27 locus (at position 12,76 Mb on chromosome 12p): D12S391 (12,34 Mb), D12S358 (12,53 Mb) and D12S1580 (13,23 Mb). We observed LOH for the 2 informative microsatellite markers near the p27 locus in the carcinoid, but not in the parathyroid adenoma (Fig. 4G).

DISCUSSION

Our current study confirms and extends previous findings concerning the presence of CDKN1B germline mutations in a sub-set of patients with multiple endocrine tumors. We identified a novel germline missense mutation in a patient with multiple endocrine and non-endocrine malignancies. Based on few recent studies the incidence of CDKN1B mutations in patients with a MEN1-related phenotype is estimated to be 1.5%-2.8% [START_REF] Georgitsi | Germline CDKN1B/p27Kip1 mutation in multiple endocrine neoplasia[END_REF][START_REF] Agarwal | Rare germline mutations in cyclin-dependent kinase inhibitor genes in multiple endocrine neoplasia type 1 and related states[END_REF]. In our patient cohort the incidence was slightly higher (1/27; 3.7%). The identification of additional mutation-positive cases will allow to more precisely assess the contribution of CDKN1B mutations to the MEN4 syndrome.

Functional in vitro characterization of the newly identified mutation (P69L), as well as of the W76X change, identified an altered molecular phenotype for both encoded proteins.

The P69L mutation affects an amino acid located in the Cdk2 binding domain of the protein (aminoacids 52-93) and this change is predicted with a high degree of confidence to reduce by more than 55% the hydrophobicity of the region (http://www.expasy.org/spdbv/), thereby dramatically altering the environmental conditions necessary for binding to the kinase. Our studies demonstrated that indeed this variant does not bind to Cdk2. The CyclinE/Cdk2 complex is a very important target of p27 and this binding is crucial for p27-mediated regulation of G1 to S phase cell cycle progression [START_REF] Sherr | CDK inhibitors: positive and negative regulators of G1-phase progression[END_REF]. Indeed, the inability of p27P69L to bind to Cdk2 associates with impaired growth suppression of GH3 neuroendocrine tumor cells in vitro.

We observed extremely reduced/absent p27 expression in the patient's tumors (parathyroid, bronchial carcinoid). This goes beyond the low expression level of p27P69L in vitro and suggests that both the wt and mutant allele are expressed at reduced level. Thus, p27P69L retains characteristics which are incompatible with sustained cell proliferation. In the carcinoid, lack of p27 expression was associated to LOH. LOH had also been observed in the tumor of the patient carrying the p27K25fs mutation [START_REF] Georgitsi | Germline CDKN1B/p27Kip1 mutation in multiple endocrine neoplasia[END_REF]. The observation that in several non-endocrine human tumors somatic biallelic inactivation of CDKN1B is exceedingly rare led to the hypothesis that p27 is a dose-dependent (haploinsufficient) tumor suppressor [START_REF] Philipp-Staheli | p27(Kip1): regulation and function of a haploinsufficient tumor suppressor and its misregulation in cancer[END_REF], as formally demonstrated in mice [START_REF] Fero | The murine gene p27Kip1 is haplo-insufficient for tumour suppression[END_REF]. In contrast, p27 might function as a canonical tumor suppressor in neuroendocrine cells. Further studies on mutation-positive patients will help clarify this issue. The patient carrying the P69L mutation shows a relatively late age at onset of endocrine disease. The few patients so far reported who carry germline CDKN1B mutations show variable age at onset. The two probands carrying the W76X or the c.59_77dup, c.K25fs mutation developed tumors at relatively young age (36 and 45 years of age, respectively), while all other carriers showed a clinical phenotype at age 50 or older. We speculate that the age at onset of the mutation-carriers might be related to how severely the CDKN1B mutation alters the p27 sequence and likely its function, which would in turn provide stronger selective advantage and lead to cell expansion.

The W76X mutation prevents the encoded p27 protein from entering the nucleus, the cell compartment where p27 exerts its function as a cell cycle inhibitor, and indeed p27W76X can no longer inhibit the growth of GH3 cells both by clonogenic assay and by stable expression. Moreover, p27W76X has lost the pro-apototic function of p27wt. This is in agreement with reports showing that cells with cytoplasmic p27 are resistant to apoptosis, but can become sensitive upon relocalization of the protein to the nucleus [START_REF] Short | AMP-activated protein kinase signaling results in cytoplasmic sequestration of p27[END_REF]. Interestingly, the truncated p27W76X protein is stable and can be detected in vitro upon transfection but also, and more significantly, in the patient's normal kidney tissue by immunohistochemistry. Although mutated alleles encoding for truncated peptides tend to be unstable and often the encoded peptides are not detected, it has been demonstrated that p27 deletion mutants comprising only the N-terminus of the protein can be expressed in vitro [START_REF] Müller | Cyclin E-mediated elimination of p27 requires its interaction with the nuclear pore-associated protein mNPAP60[END_REF][START_REF] Fuster | Tumor suppressor p27Kip1 undergoes endolysosomal degradation through its interaction with sorting nexin 6[END_REF]. Although the W76X mutation alters several p27 functions, the only tumor tissue of a mutation-positive patient available for analysis showed no p27 immunoreactivity (Pellegata et al., 2006) while the truncated protein was present in the patient's normal tissue. This indicates that this mutation possesses functions that are detrimental to, or no longer needed by, neuroendocrine tumor cells and therefore selected against during tumor progression. These functions might affect still unidentified mechanisms of p27 action and deserve further evaluation.

Studies on naturally-occurring CDKN1B mutations will help us understand the link between p27 and neuroendocrine tumor predisposition, a prerequisite to offer in the future personalized care to the families carrying such mutations.

SUPPLEMENTAL MATERIALS AND METHODS

DNA analysis

Patient DNA from blood and formalin-fixed, paraffin-embedded (FFPE) tissue blocks was extracted and sequenced as already reported (Pellegata et al. 2006). Unrelated control DNA samples were obtained from the Institute of Human Genetics, Helmholtz Zentrum München and comprise 380 healthy Germans (KORA Study Group). Loss of heterozygosity (LOH) analysis was performed as previously reported (Shyla et al 2010) using the chromosome 12p markers D12S391, D12S358, and D12S1580.

Plasmid constructs and antibodies

The two described mutations were introduced by site-directed mutagenesis (Quikchange II Site-Directed Mutagenesis Kit, Stratagene) in the wild-type human CDKN1B cDNA cloned in a pEYFP backbone, kindly provided by J. Slingerland (University of Miami, FL).

Primary antibodies used were: anti-p27 (clone 57) and anti-GFP tag (BD Biosciences); anti-Skp2 (H-345); anti-Cdk2 (D-12); anti-Cdk4 (C-22); anti-Cyclin D1 (H-295); anti-Cyclin E (M-20); α-tubulin (all from Santa Cruz Biotechnology).

Cell culture, transfections, drug treatments

HeLa and MCF7 cell lines were respectively maintained in DMEM or RPMI 1640 media supplemented with 10% fetal bovine serum, 20 mM L-glutamine, 100 units/ml of penicillin G sodium, and 100 µg/ml streptomycin (all Invitrogen). The GH3 cell line was maintained in F-12K medium supplemented with 15% horse serum, 2.5% fetal bovine serum, 20 mM L-glutamine, 100 units/ml of penicillin G sodium, and 100 µg/ml streptomycin.

For p27 half-life determination in exponentially growing cells, cycloheximide (CHX, Sigma-Aldrich, 25 µg/ml) was added to the cells for the indicated times.

Transfections were carried out as already reported (Pellegata et al. 2006). When establishing stable transfectants, GH3 cells were selected by adding 0.4 mg/ml G418 to the culture medium for 2-3 weeks and individual colonies picked and expanded in presence of G418.

RNA interference

For RNAi studies, short interfering RNA (siRNA) duplexes specific for SKP2 (n=4; siGenome SMARTpool, Dharmacon) and KPC1 (n=2; ID 133486, 133487 Ambion) and scrambled siRNA (Ambion) were obtained and transfected into MCF7 cells using X-tremeGENE reagent (Roche) together with 0.03 µg of plasmid DNA.

Protein extraction, immunoblotting and immunoprecipitations

Protein extraction and immunoblotting were carried out as already reported (Pellegata et al. 2006).

For IP, 500 µg of total protein were incubated with 5 µl of anti-YFP tag antibody (Clontech-Takara Bio Europe) and 20 µl of agarose resin (Santa Cruz) overnight at 4 °C. For GST-pull down assay, 500 µg of total protein were incubated overnight at 4° with 2 µg of recombinant Cdk2-GST (Abnova) and 20 µl of Glutathione Agarose (Santa Cruz).

Intracellular localization and apoptosis assay

p27 knock-out (-/-) MEFs were obtained from 13 days old embryos upon trypsin digestion for 15 min at 37ºC. Cells were grown in DMEM supplemented with 10% fetal bovine serum, 20 mM Lglutamine, 100 units/ml of penicillin G sodium, and 100 µg/ml streptomycin (all Invitrogen). For localization experiments, MEFs were grown on coverslips, transfected and processed for immunocytochemistry as described (Pellegata et al. 2006) with minor modifications. For the apoptosis assay, HeLa cells were transfected while exponentially growing on coverslips. At 24h or 48h posttransfection cells were fixed and nuclei were stained with DAPI.

Clonogenic assay and growth curve

To examine clonogenic activity, GH3 cells were plated (500,000 per well) in 6-well plates and transfected with p27wt, p27W76X or p27P69L using Fugene-6 (Roche). The day after the transfection, the cells were diluted 1:6 and, starting from the day after, selected for 6 weeks by adding 0.4 mg/ml G418 to the culture medium and stained with 0.3% crystal violet in 30% ethanol. Colonies that contained 50 cells were scored. p27W76X GH3 stable clones, and untrasfected GH3 cells were plated in duplicate (100,000 per well) in 12-well plate, in full medium plus 400 µg/ml G418. The number of cell was counted every two days over a 9 day-period using a cell counter.

Immunohistochemistry

Immunohistochemistry was performed using a monoclonal anti-p27 antibody (BD Biosciences) as already reported (Pellegata et al. 2006).

Figure 1

 1 Figure 1Characterization of the P69L and W76X mutations in p27. A, Sequence chromatograms showing the novel nucleotide change identified in patient FL compared to a control normal individual. B, Expression of the p27W76X and p27P69L mutant proteins 24h after transfection of MCF7 cells. Western blotting (WB) was performed with a monoclonal anti-p27 antibody. C, Localization of the p27 proteins. Asynchronously growing p27-negative MEFs were transiently transfected with the different YFP fusion constructs. Cells were fixed 24h later and nuclei were counterstained with DAPI. Cells were examined for direct YFP fluorescence. D, p27P69L does not associate with Cdk2. Lysates obtained upon transfection of HeLa with p27wt or p27P69L were incubated with purified GST-Cdk2 protein. Following GST pull-down, 50 µgr of the original lysate (Input) and the entire pull-down (GST-Cdk2) were analyzed by Western blotting (WB) with antibodies against p27 or Cdk2.

  Colonies were stained and counted. The values represent the mean of 2 independent experiments (each consisting of 6 separate plates) ±SD. C, Growth curves of representative GH3derived clones expressing p27W76X. At day 1 and day 9 cells were lysed and total proteins were analyzed by Western blotting (WB). Values are the mean of 2 independent experiments ±SD. Only the transfected proteins are shown. D, p27wt, p27W76X and p27P69L fusion proteins were transiently transfected into HeLa cells and 48h later stained with DAPI. Cells positive for YFP (=transfected) and showing chromatin condensation (=apoptotic) were counted and expressed as the percentage of the YFP-positive cells without features of apoptosis (Y axis). The values represent the average of 6 independently-transfected plates ±SD.

  Figure 4 Expression of p27P69L and p27W76X in patient tissues. A-F, Immunohistochemical staining with an anti-p27 specific antibody. A-B, Staining of the normal kidney tissue from the W76X mutation-positive patient who developed renal angiomyolipoma (A), and from a normal control individual (B). The patient with the germline p27 mutation shows cytoplasmic staining in the renal tubules, whereas the normal individual only shows nuclear p27 immunoreactivity in some tubules. C-D, Staining of the parathyroid tumor from the P69L mutation-positive patient (C) and of a similar tumor from a sporadic mutationnegative patient (D). The p27 immunoreactivity of the tumor cells in the mutation-positive patient is dramatically reduced compared to the interspersed endothelial cells (indicated by arrows in the Inset). E-F, Staining of one bronchial carcinoid from the P69L mutation-positive patient (E) and of a similar tumor from a sporadic patient without mutation in p27 (F). There virtually lack of p27 immunoreactivity of the tumor cells of the mutation-positive patient compared to the interspersed endothelial cells (indicated by an arrow in the Inset). Bar, 50 µM. Original magnification: A-F, 200X; Insets, 400X. G, LOH analysis of the tissues of the P69L mutation-positive patient for 3 microsatellite markers adjacent to the p27 locus. PT, parathyroid tumor; Carc N, normal tissue adjacent to the bronchial carcinoid; Carc T, bronchial carcinoid (tumor tissue).

John Wiley & Sons, Inc.Human Mutation

ACKNOWLEDGEMENTS

We thank the patients for their kind participation, Dr. G. Keller and Prof. T. Illig for genotyping, Dr. V. d'Agostino for help with the protein analysis software.

This work was in part supported by grant # 107973 from the Deutsche Krebshilfe (N.S.P.). Dr. S. Molatore was the recipient of a post-doctoral fellowship of the Centro Comunicazione e Ricerca -Collegio Ghislieri, Pavia, Italy.

Contract grant sponsor: Deutsche Krebshilfe; Contract grant number: 107973.