Anne-Laure Le Nadant
email: anne-laure.lenadant@unicaen.fr

Frédéric Perdreau
email: frederic.perdreau@univ-st-etienne.fr

LBOs and innovation: the French case

Keywords: Buyouts, Innovation, Private Equity Firms

A long-standing controversy is whether LBOs generate economic efficiencies through a superior governance framework, or whether LBO funds are driven by short-term profit motives and sacrifice long-term growth to boost short-term performance. Using a propensity score methodology, this paper provides an empirical analysis of the innovative efforts of a sample of 89 French manufacturing firms that underwent a buyout between 2001 and 2005. The matching estimates (average treatment on the treated, ATT) of the effect of LBOs on firm level of innovation expenditures in 2006 show no significant differences between LBO targets and comparable companies that did not go through an LBO. In contrast, we find significant effects of LBOs on both service innovation and marketing innovations in design and packaging and product promotion.

Introduction

Innovation is an essential preoccupation for a firm as it affects its competitiveness. A longstanding controversy is whether LBOs generate economic efficiencies through a superior governance framework, or whether LBO funds are driven by short-term profit motives and sacrifice long-term growth to boost short-term performance.

LBOs involve investments in which investors and a management team pool their own money (together with debt finance) to buy shares in a target company from its current owners [START_REF] Meuleman | Agency, Strategic Entrepreneurship, and the Performance of Private Equity-Backed Buyouts[END_REF]. Although the change in governance resulting from LBOs is generally found to exert a positive impact on firm"s economic and financial performance [START_REF] Cumming | Private equity, leveraged buyouts and governance[END_REF], these transactions have mainly been associated with cost-cutting activities and shorttermism, to the detriment of innovation and Research and Development (R&D) investments. However, in a recent study, [START_REF] Boucly | Growth LBOs[END_REF] show that, instead of reinforcing credit constraints, as was the case in the 1980s transactions, today"s LBOs can alleviate them.

Whether LBOs have an impact on innovation is not clear, a priori. Both positive and negative effects are likely to occur. After an LBO, technological matters may be delayed or set aside because managers may be more oriented to day to day operations resulting from the transaction [START_REF] Hitt | The Market for Corporate Control and Firm Innovation[END_REF] or because private equity firms exert pressure on management to focus on investment opportunities that are less uncertain and more rewarding in the short term [START_REF] Ughetto | Assessing the contribution to innovation of private equity investors: a study on European buyouts[END_REF].

Alternatively, LBOs may foster entrepreneurial initiatives, enabling managers to better and more thoroughly exploit firm resources for new innovation projects (Wright, Hoskisson and Busenitz, 2001). There are two reasons for this. First, private equity-backed buyouts can make use of the private equity firm's extensive network and relationships: customers, suppliers, other investors, access to more sophisticated resources in banking, legal and other areas, etc. Second, the private equity firm's expertise and competencies with regard to strategy, operational and financial management, human resources, marketing policy, and mergers and acquisitions, also help create value for the target firm [START_REF] Lee | Internal capabilities, external networks, and performance: A study on technology-based ventures[END_REF][START_REF] Wright | Finance and management buyouts: Agency versus entrepreneurship perspectives[END_REF]. Inside management does not always own the tacit knowledge and idiosyncratic skills required to seize new opportunities [START_REF] Hendry | The principal's other problems: Honest incompetence and the specification of objectives[END_REF]. If major innovation is required, it may be necessary to introduce outside managers who do own these skills (Wright, Hoskisson, and Busenitz, 2001). In this situation, the private equity firm plays an important role in assessing the skills of the incumbent managers and their potential replacements [START_REF] Meuleman | Agency, Strategic Entrepreneurship, and the Performance of Private Equity-Backed Buyouts[END_REF].

Evidence regarding the impact of LBOs on investment in innovation and R&D is so far limited and rather mixed. Some studies show a decline in R&D spending [START_REF] Long | LBOs, Debt and R&D Intensity[END_REF] whereas others find no decline [START_REF] Lichtenberg | The effects of leveraged buyouts on productivity and related aspects of firm behavior[END_REF] or stability [START_REF] Smith | Corporate Ownership Structure and Performance: the Case of Management Buyouts[END_REF] of research spending after the LBO. However, as most firms involved in LBOs do not belong to technology-intensive industries, the impact of LBOs on cumulative innovation is likely slight [START_REF] Hall | The impact of corporate restructuring on industrial research and development[END_REF].

In industries where R&D requirements are more crucial, these expenditures are used more effectively. [START_REF] Zahra | Corporate Entrepreneurship and Financial Performance: The Case of Management Leveraged Buy-Outs[END_REF] finds, on a sample of 47 LBOs, that firms involved develop more new products and intensify their efforts in terms of innovation and productivity (even if at the same time the level of R&D expenditure doesn"t change). Wright et al. (2001) provide several examples of buyouts in technology-based industries followed by significant increases in product and technology development, R&D and patenting. [START_REF] Malone | Characteristics of smaller company leveraged buyouts[END_REF] and [START_REF] Wright | Venture capital and management-led, leveraged buy-outs: A European perspective[END_REF] also cite evidence of new product innovation following buyouts.

More recently, [START_REF] Lerner | Private Equity and Long-Run Investment: The Case of Innovation[END_REF] investigate 472 LBOs with a focus on investments in innovation as measured by patenting activity. They find no evidence that LBOs are associated with a decrease in these activities. Contrary to the frequent argument that private equity firms have short-term horizon and little incentive to favor long-term investment opportunities of target companies, this study shows that LBOs lead in fact to significant increases in long-term innovation. The authors find that patents granted to firms involved in LBOs are more cited (a proxy for economic importance) and show no significant shifts in the fundamental nature of the research. Hence, if some US and UK based studies show a decline in investment expenditure after LBOs, critical investments in R&D seem to be maintained. [START_REF] Ughetto | Assessing the contribution to innovation of private equity investors: a study on European buyouts[END_REF] has focused on innovation of Western European manufacturing firms undergoing an LBO. She finds that innovation activity of portfolio firms (measured by the number of patents granted) is affected by different types of investors, pursuing different objectives.

There are a several reasons to revisit the questions in the previous studies. First, the private equity industry is more substantial today than it was in the 1980s. Changes in the industry-such as the increased competition between and greater operational orientation of private equity firms-suggest that the earlier relationships may no longer hold. Second, transactions involving technologyintensive industries have become more common recently and it is also desirable to look beyond public-to-private transactions, since these transactions represent a fairly small fraction of the private equity universe. Third, the innovation surveys conducted in the last two decades have substantially enhanced our ability to measure and study the impact on innovation. Fourth, nearly all studies on LBOs and innovation have concentrated on the US and the UK (the only exception we are aware of is the study of Western Europe by [START_REF] Ughetto | Assessing the contribution to innovation of private equity investors: a study on European buyouts[END_REF]. By shifting the focus to France and following [START_REF] Boucly | Growth LBOs[END_REF], this paper investigates the possibility that some LBOs aim to seize innovation opportunities and expand the scale and scope of the target"s activities.

France is an interesting context to study LBOs and innovation because it is a country with many family-managed firms that tend to be, on average, smaller than non family firms and for which access to external finance may be more difficult than in the US or the UK.

The remainder of the paper is as follows. In Section 2, we describe the construction of the sample. Section 3 reviews the methodology employed in the study. We present the empirical analyses in Section 4. The final section concludes the paper and discusses future work.

Dataset

To analyze the impact of LBOs on innovation at the company level, we use a new database built from three different databases: Capital IQ (to isolate transactions), CIS 2006 (for innovation data) and DIANE (for financial statements). To our knowledge, this is the first study to use a CIS survey (Community Innovation Survey) in relation with LBOs. Community Innovation Surveys are conducted at regular intervals in Europe. Questions are based on the Oslo Manual guidelines, which distinguishes four types of innovations: product innovations, process innovations, organizational innovations and marketing innovations. The Oslo Manual opted for collecting data at the firm level, including all its innovation outputs and activities, which is also the level of available accounting and financial data that can be merged with the innovation data for richer analyses. CIS 2006 was launched in 2007, based on the reference period 2006, with the observation period 2004 to 2006. The population of the CIS is determined by the size of the enterprise and its principal activity. All enterprises with 20 or more employees in any of the specified sectors were included in the statistical population. The following industries were included in the population of the CIS 2006: mining and quarrying (NACE 10-14), manufacturing (NACE 15-37), electricity, gas and water supply . Three reference periods were used in the questionnaire:

 The first relates to a set of questions for the whole of the period 2004-2006, for example whether the enterprise introduced an innovation at any time during this three-year period.

 The second set of questions refers uniquely to the reference year 2006, for example, indicators such as innovation expenditure.

 Finally, a limited number of basic economic indicators were requested for both 2004 and 2006, for example the turnover and employment figures.

We first identify 944 French deals over 2001-2005 reported as being "LBOs" from Capital IQ. More precisely, we retrieve all the deals from Capital IQ with the following characteristics: (i) they are announced between 2001 and 2005 (ii) either "closed" or "effective" (iii) reported by Capital IQ as being "LBOs". Most of the targets are medium sized, privately held firms. We then obtain innovation data from CIS 2006. Our transaction and innovation data do not have the same identifier so we match them by company name. Names are not always identical in both databases, so in case of ambiguity we resort to company websites and annual reports. The matching process reduces sample size to 109 transactions, of which 89 have available financial statements in Diane (Bureau Van Dijk) for the year preceding the transaction.

Table 1 summarizes the characteristics of the final sample. 47% of deals take place since 2004. Slightly less than 40% of the sample is composed of companies in intermediate goods (52% in terms of value of the deals). In terms of size, the sample is mostly constituted of relatively small companies: 34% of targets have less than 20 million (M) Euros in sales at the time of the deal, and 67% have less than 75M. Companies with sales above 75M constitute 31% of number but 73% of the value of deals.

Methodology

Gauging effects of LBOs on innovation is not trivial because LBOs do not occur randomly across the population of firms. LBO targets are selected by investors presumably because of their value creation potential. If an outside observer concludes that the average level of innovation expenditure of firms targeted by an LBO is higher than in other firms, one cannot rule out the possibility that this finding is due to the fact that LBO investors tend to select better firms on average relative to the population.

We address the problem of sample selection bias using a propensity score methodology (PSM) [START_REF] Rubin | Estimating causal effects of treatments in randomized and non randomized studies[END_REF][START_REF] Heckman | The economics and econometrics of active labour market programs[END_REF]. We benchmark the level of innovation of LBO firms by selecting appropriate matching control firms to each LBO. The set of matching control firms is composed of firms that share the same financial characteristics as the LBO firm prior to the transaction. To select matching firms that have ex-ante the same probability of being selected by LBO investors, we implement a probit model to estimate the likelihood of being an LBO target in a given year and we use the use the probability estimate from that model to find a matching control for a firm that indeed was the target of an LBO deal1 .

The main steps of the PSM procedure are as follows. First, we introduce filters to obtain a dataset composed of about 1,200 companies. We need to do this because fitting a discrete choice regression model where the number of "zeros" (that is, observations where the firm is not an LBO target in a given year) is very high relative to the number of "ones" (that is, observations where the firm is an LBO target in a given year) results in poor estimates. This is the case since CIS 2006 contains data for about 5,200 companies, and the sample contains 89 LBOs (2 % of the dataset). We therefore introduce filters to obtain a manageable number of non-LBO observations. A matching company belongs to the same 4-digit sector as the target. If there are more than ten twins, we just keep the ten nearest neighbors to the target with the nearest turnover the year preceding the buyout. The matching methodology allows us to retain 1,144 "twin" companies to the sample, i.e. 12.85 twins by target. We choose 1,200 as a number that seems reasonable because it means that LBOs constitute about 8% of the regression sample.

Second, we run a probit regression that models the likelihood of a firm being the target of an LBO in a particular year. Denote by h k * the latent unobservable variable that represents the net present value of the acquisition of firm k by a bidder and h k,t a dummy that takes the value of 1 if an LBO bid is made in year t: h k,t = 1 if h k,t * >0 or h k,t = 0 if h k,t * < 0. The probit regression to be estimated for the probability of Pr(h k,t = 1) is:

h k,t * = α +W k,t δ + υ k,t (1)
The matrix W k,t contains firm-specific variables that the literature has identified as determinants of the likelihood that a firm is an LBO target, namely firm size (measured by turnover), the debtequity ratio, the level of income taxes, the firm"s profitability (measured by ROIC), liquidity (proxied by cash divided by assets) and level of working capital (Le [START_REF] Nadant | Financial Profile of Leveraged Buyout Targets: Some French Evidence[END_REF].

The predicted value from regression model ( 1) is called the propensity score. Its interpretation is that it measures the probability, as predicted by the model, that a firm becomes an LBO target in a given year. In other words, firms with similar propensity scores share similar characteristics that lead to being an LBO target. They constitute therefore adequate benchmarks for LBO innovation capacity.

Third, we use propensity score to match comparison units with treated units. Smith and Todd (2005) note that measuring the proximity of cases as the absolute difference in the propensity score is not an approach that is robust to "choice-based sampling," where the treated are oversampled relative to their frequency in the population of eligible individuals [START_REF] Caliendo | Some Practical Guidance For The Implementation Of Propensity Score Matching[END_REF]. As a consequence, we match on the log odds of the propensity score, defined as p/(1-p), to assure that results are invariant to choice-based sampling.

Different matching algorithms can be used: kernel matching, nearest-neighbor or radius matching. As we have many firms not involved in LBOs in our dataset, the radius matching algorithm (with a caliper of 0.06) is more appropriate: it enables us to compare firms with very close predictions of probit models. To avoid the risk of poor matches, radius matching specifies a "caliper" or maximum propensity score distance by which a match can be made. The basic idea of radius matching is that it uses not only the nearest neighbor within each caliper, but all of the comparison group members within the caliper. In other words, it uses as many comparison cases as are available within the caliper, but not those that are poor matches (based on the specified distance). The robustness of our results is tested using other matching methods. We then measure, for each LBO, its level of innovation relative to the level of innovation of its matching control pair.

Post LBO innovation: evidence and robustness a) Main results

Table 2 presents summary statistics for accounting measures of LBOs and the non-LBOs firms. Relative to their potential controls, LBO firms are larger (65 M in average turnover vs. 40M) and slightly more profitable (23% vs. 20% in terms of average ROIC), and they have higher income taxes expenses the year before the deal (2.39% of sales for LBO vs. 1.63% for non-LBO). Only differences in size and income taxes expenses are significant (see Appendix 2).

Table 2 -Summary statistics

This table shows the summary statistics for sample deals for the year before the deal. «LBO companies» refers to statistics of the sample of LBO firms. «Non-LBO companies» refers to statistics of the sample of all non-LBO companies from which matched controls are chosen using a propensity score model. All these accounting variables are obtained from DIANE. Turnover is in thousands of Euros. Income taxes expense and working capital are divided by turnover. Working Capital is divided by net fixed assets. Debt-to-equity is measured by financial debt divided by shareholders" equity (in %).

Variable

Obs The results of the probit model show that the largest firms and those that have the highest level of income taxes have a higher probability of being an LBO target (Table 3). In contrast, firms" financial structure, profitability, liquidity and level of working capital do not seem to explain LBO likelihood. The matching estimates (average treatment on the treated, ATT) of the effect of LBOs on firm level of innovation in 2006 show no significant differences between LBO targets and comparable companies that did not go through an LBO. Moreover, we find no significant effect of LBOs on innovation expenditure at different periods of time (one-year after transaction for LBOs in 2005, two-year after transaction for LBOs in 2004, etc.). Evidence thus suggests that private equity intervention is not detrimental to long-term investments in innovation.

Table 4 -Effect of LBOs on the level of innovation expenditure in 2006

Innovation expenditure is measured by all innovation expenditure in 2006 declared by firms as a percentage of turnover the same year.

Variable

Number of observations 89 1144

Notes: Significance levels: ***(1%), **(5%) and *(10%).

Even if LBO targets have the same level of innovation expenditure as their matched counterparts, LBOs might have an effect on other innovation outcomes. Indeed, LBOs might foster entrepreneurial initiatives and innovation projects that are not costly and do not require important expenditure. Table 5 presents PSM estimations on innovation outcomes for LBOs that occurred from 2001 to 2004 (69 LBOs) whereas Appendix 1 presents the innovation outcomes and innovation variables definitions. As questions in CIS 2006 relate to the 2004-2006 period, we exclude 20 LBOs that occurred in 2005 from the sample to run these estimates. The comparison of unmatched and ATT estimates (not reported here) shows the efficiency of the matching method, which significantly reduces the selection bias. Indeed, differences between LBOs and non-LBOs on several outcome variables appear to be significant when they are estimated with unmatched units. For instance, for product innovation (inpdgd) and innovation in external relations (oorgexr), t-statistic values are respectively 1.68 and 1.78 (significant at 10% level) but, after matching, the differences are no longer statistically significant, suggesting that matching helps reduce the bias associated with observable characteristics.

Table in Appendix 2 shows the results of the balancing test for the radius model. It confirms the validity of the matching method, which significantly reduces the bias for all covariates. The balancing test measures the "similarity" of observations remaining after the matching process. Regarding product and process innovations, we find that LBO targets are more likely to implement service innovations. Hence, results suggest that LBOs lead to significant improvements in how services are provided (for example, in terms of their efficiency or speed), the addition of new functions or characteristics to existing services, or the introduction of entirely new services. However, as we also find an effect on innovations that are new to the firm but no effect on innovations that are new to the market, we can conclude that LBOs lead to minor product innovations that have already been implemented by other firms. Hence, it is likely that the nature of the innovation in LBOs is based upon incrementally improving current products for existing markets rather than developing new products and processes [START_REF] Zahra | Will leveraged buyouts kill U.S. corporate research and development?[END_REF].

Regarding organisational and marketing innovations, we find significant effects for two variables of marketing innovation only: innovations in design and packaging and in products promotion. Marketing innovations are aimed at better addressing customer needs, opening up new markets, or newly positioning a firm"s product on the market, with the objective of increasing the firm"s turnover. LBOs have a significant effect on changes in product design and packaging that are intended to change or enhance the product"s appeal or to target a new market or market segment. They also have a positive impact on promotional efforts made by firms to improve their products" image or to increase awareness of their products. This evidence confirms that private equity firms provide superior managerial and technical expertise that enables target firms to seize innovative opportunities.

In contrast, we find no effect of LBOs on the methods of protecting innovations. In particular, we show that LBOs have no impact on patenting activity. This result is in line with [START_REF] Lerner | Private Equity and Long-Run Investment: The Case of Innovation[END_REF].

Last, innovation activity may be hampered by a number of factors. There may be reasons for not starting innovation activities at all, or factors that slow innovation activity or have a negative effect on expected results. These include economic factors, such as high costs or lack of demand, and enterprise factors, such as a lack of skilled personnel or knowledge. Regarding obstacles to innovation, results show an effect of LBOs on two factors hampering innovation activities: lack of skilled personnel and lack of external financial resource. These results suggest that LBO targets do not have the skilled personnel needed to engage in innovation activities, or their innovation activities may be slowed because they are unable to find the necessary personnel on the labour market. Moreover, contrary to [START_REF] Boucly | Growth LBOs[END_REF] who show that LBOs can alleviate financial constraints and foster growth, our results suggest that LBO targets lack external financial resource for their innovation activities.

b) Robustness check

We evaluate the robustness of the estimations by changing the matching algorithms. The matching algorithms used are nearest neighbor (with one and three neighbors), radius (with a caliper of 0.06) and kernel. The impact on innovation variables does not appear to depend critically on the algorithm used, since both the value of the coefficients and its significance are very similar using different alternatives. In particular, we find no impact of LBOs on innovation expenditure whatever the algorithm used.

Conclusion

Hence, although most commentators in the public debate and many financial economists consider LBOs as a way to implement drastic, "cost cutting" measures, this paper provides evidence that ex-post innovation expenditure is similar LBO targets and comparable firms in France. In addition, we find no evidence that, as for contribution to innovation, LBOs alleviate financial constraints or facilitate business relations.

In contrast, we find an effect of LBOs on service innovation but this effect is not related to radical or disruptive innovation as LBOs have an impact on product innovation that is new only to the firm. Moreover, results show that LBO targets are more likely to implement marketing innovations (design and packaging and product promotion) in order to increase turnover and market share. This result is in line with the idea that, following an LBO, management is not only focused on "cost cutting" activities that aim to increase value creation. Private equity firms also encourage managers of LBO targets to build new strategies to find and exploit value creation potentials. Further, these results emphasize the resources and capabilities that buyout specialists bring in terms of contribution to innovation to their portfolio companies as they suggest that LBOs do not lead to the acquisition of skills or resources that enable the introduction of disruptive innovation. Adversely, results show that post-LBO innovations, which are mainly based upon marketing and service innovations, rely on existing resources with only the addition of a marketing competency.

Contrary to [START_REF] Boucly | Growth LBOs[END_REF], results also suggest that LBOs do not alleviate financial constraints as we find a positive effect of LBOs on the lack of financial resources as a factor hampering innovation. Moreover, as we find no effect of LBOs on radical innovations or new products or services, it seems that LBOs do not help to achieve such radical innovation. This result could be a clue that LBOs lead to the strengthening of financial and human resources constraints that hamper radical innovation.

Table 1 -Descriptive statistics of final sample

 1 This table shows the number and value of deals in the sample. Value is measured using the sum of sales revenue of companies in each category, in thousands of Euros, for the year prior to the deal. Breakdown by sector follows the French classification named NES16 (Nomenclature Economique de Synthèse).

	Panel A : Breakdown by year
		Number	%	Value	%
	2001	18	20.22%	1 099 628	19.07%
	2002	12	13.48%	1 000 782	17.35%
	2003	17	19.10%	1 139 305	19.76%
	2004	22	24.72%	1 162 113	20.15%
	2005	20	22.47%	1 364 967	23.67%
	Total	89		5 766 795
	Panel B : Breakdown by Sector
		Number	%	Value	%
	Agricultural and food industries	7	7.87%	511 133	8.86%
	Consumer Goods Industry	13	14.61%	596 591	10.35%
	Automotive Industry	6	6.74%	241 696	4.19%
	Capital goods industries	24	26.97%	1 232 990	21.38%
	Intermediate goods	35	39.33%	3 019 754	52.36%
	Energy	4	4.49%	164 630	2.85%
	Total	89		5 766 795
	Panel C : Sample Breakdown by Sales revenues
	(Sales in Thousand of Euros)	Number	%	Value	%
	(0;20000]	31	34.83%	303046	5.26%
	(20000;75000]	30	33.71%	1201816	20.84%
	(75000;150000]	18	20.22%	1762976	30.57%
	(150000; max]	10	11.24%	2498958	43.33%
	Total	89		5 766 795

Table 3 -Estimations of the Probit model

 3 Turnover is in thousands of Euros. Income taxes and working capital variables are divided by turnover. Liquidity variable is measured by cash and cash equivalents divided by total assets. Debt-to-equity is financial debt divided by shareholders" equity.

		Probit regression on 2001-2004	Probit regression on 2001-2005
	Indep.variables	Coef	Std. Error	Coef	Std. Error
	Turnover	0.1278***	0.0427	0.1521***	0.0389
	ROIC	0.0629	0.1063	0.0234	0.1040
	Debt-to-equity	-0.0000	0.0001	-0.0000	0.0001
	Income taxes	6.2601***	2.3278	4.3494**	1.9705
	Liquidity	-0.0103	0.0296	-0.0267	0.0554
	Working capital	0.0698	0.1245	0.0744	0.1171
	Constant	-3.7741***	0.7318	-4.1082***	0.6705
		Number of Obs. = 1004 (69 LBOs)	Number of Obs. = 1233 (89 LBOs)
		LR Chi2(6) = 18.31		LR Chi2(6) = 23.01
		Prob > Chi2 = 0.005		Prob > Chi2 = 0.001
		Pseudo R2 = 0.036		Pseudo R2 = 0.036
	Notes: Significance levels: ***(1%) and **(5%).

Table 4

 4 reports both unmatched and matching estimates (average treatment on the treated, ATT) of the effect of LBOs on firm level of innovation, measured as innovation expenditure in 2006 divided by turnover in 2006.

Table 5 -ATT estimates of innovation outcomes during 2004-2006

 5

	Variable type	Variable code	Treated	Controls	Difference	S.E.
		inpdgd	.5147	.4385	.0761	.0632
		inpdsv	.2500	.1594	.0905*	.0542
		newmkt	.4264	.3249	.1014	.0623
	Product innovations	newfrm	.4411	.3237	.1174*	.0626
		turnmar	.0613	.0645	-.0031	.0197
		turnin	.0544	.0483	.0060	.0172
		turnung	.8842	.8871	-.0028	.0277
		inpspd	.3676	.3704	-.0027	.0610
	Process innovations	inpslg	.1911	.1619	.02918	.0495
		inpssu	.1764	.1718	.0045	.0482
		oorgbup	.3823	.3905	-.0081	.0615
	Organisational	oorgkms	.2205	.2517	-.0312	.0527
	innovations	oorgwkp	.3088	.3183	-.0095	.0585
		oorgexr	.2500	.1762	.0737	.0544
		mktdgp	.3235	.1869	.1365**	.0585
	Marketing	mktpdp	.2647	.1686	.0960*	.0553
	innovations	mktpdl	.1029	.0988	.0040	.0383
		mktpri	.1617	.1044	.0573	.0461
		ProPat	.2941	.3245	-.0304	.0577
		ProDsg	.2352	.1954	.0398	.0534
		ProTm	.3823	.2847	.0976	.0611
	Patents and other	ProCp	.1029	.0676	.0353	.0380
	protection
	methods	prosol	.1029	.0666	.0363	.0380
		ProSct	.1764	.1479	.0285	.0479
		ProCon	.2647	.2407	.0239	.0557
		ProTech	.2352	.1776	.0576	.0533

For a binary treatment variable, there is no strong advantage to using the logit vs. probit model. We also ran a logit model and found similar results.


Christel Depienne

Eric Leguern

PCDH19-related Infantile Epileptic Encephalopathy: An Unusual X-linked Inheritance Disorder

Keywords: PCDH19, Epilepsy, X-linked, females, male sparing, cellular interference

PCDH19 encodes protocadherin 19 on chromosome Xq22.3. This 1148 amino-acid protein, highly expressed during brain development, could play significant roles in neuronal migration or establishment of synaptic connections. PCDH19 is composed of 6 exons, with a large first exon encoding the entire extracellular domain of the protein. Heterozygous PCDH19 mutations were initially identified in epilepsy and mental retardation limited to females, a familial disorder with a singular mode of inheritance since only heterozygous females are affected whereas hemizygous males are asymptomatic. Yet, mosaic males can also be affected, supporting cellular interference as the pathogenic mechanism. Recently, mutations in PCDH19, mostly occurring de novo, were shown to be a frequent cause of sporadic infantile-onset epileptic encephalopathy in females.

PCDH19 mutations were also identified in epileptic females without cognitive impairment.

Typical features of this new epileptic syndrome include generalized or focal seizures highly sensitive to fever, and brief seizures occurring in clusters, repeating during several days. Here we present a review of the published mutations in the PCDH19 gene to date and report on new mutations. PCDH19 has become the second most relevant gene in epilepsy after SCN1A.

INTRODUCTION

Mutations in PCDH19 (MIM# 300460) were originally identified in 2008 in rare large families in which female patients had variable degrees of epilepsy and intellectual deficits. This disorder, first reported as epilepsy and mental retardation limited to females (or epilepsy, femalerestricted, with mental retardation, EFMR) and recently renamed Epileptic encephalopathy, early infantile, 9 (MIM# 300088), was transmitted via asymptomatic males, suggesting an unusual Xlinked inheritance with selective involvement of females [START_REF] Juberg | A new familial form of convulsive disorder and mental retardation limited to females[END_REF][START_REF] Fabisiak | A familial form of convulsive disorder with or without mental retardation limited to females: extension of a pedigree limits possible genetic mechanisms[END_REF][START_REF] Ryan | Epilepsy and mental retardation limited to females: an X-linked dominant disorder with male sparing[END_REF][START_REF] Dibbens | X-linked protocadherin 19 mutations cause female-limited epilepsy and cognitive impairment[END_REF].

Recently, PCDH19-related infantile epileptic encephalopathy turned out to be more frequent than first anticipated on the observation of families, PCDH19 becoming the second most clinically relevant gene in epilepsy after SCN1A (MIM# 182389). Most patients with PCDH19 mutations are sporadic cases or belong to families with few affected female patients, making the recognition of the inheritance pattern difficult. In addition, associated clinical features, including age at onset, seizure types and severity, and the degree of cognitive disability, are highly variable. However, clinical characteristics such as clustering of repeated seizures within short periods of time are now emerging as hallmark features that orient etiological diagnosis towards PCDH19. This mutation update summarizes the mutations identified in the gene and the recent findings in the field.

Background

Protocadherins (pcdhs) are transmembrane proteins primarily involved in calcium-dependent adhesion that constitute the largest subgroup of the cadherin superfamily. Mammalian genomes contain more than 70 Pcdh genes that are divided into two groups based on their genomic structure: clustered (58 genes, Pcdh, , ) and non-clustered Pcdh genes (13 genes, Pcdh and other Pcdhs). Pcdhs typically have six or more extracellular calcium-binding cadherin repeats or ectodomains (EC domains), which are required for cell-cell homophilic or heterophilic interactions, and a divergent cytoplasmic domain [START_REF] Yagi | Cadherin superfamily genes: functions, genomic organization, and neurologic diversity[END_REF][START_REF] Patel | Cadherin-mediated cell-cell adhesion: sticking together as a family[END_REF][START_REF] Redies | delta-Protocadherins: unique structures and functions[END_REF][START_REF] Patel | Type II cadherin ectodomain structures: implications for classical cadherin specificity[END_REF][START_REF] Morishita | Protocadherin family: diversity, structure, and function[END_REF]. Protocadherins, like many other cadherins, are predominantly expressed in the brain, where they play significant roles in neurodevelopment, such as neuronal migration and synaptic plasticity [START_REF] Frank | Protocadherins[END_REF][START_REF] Junghans | Mammalian cadherins and protocadherins: about cell death, synapses and processing[END_REF][START_REF] Redies | delta-Protocadherins: unique structures and functions[END_REF][START_REF] Morishita | Protocadherin family: diversity, structure, and function[END_REF]. The combinatorial expression of multiple cadherin and protocadherin genes could contribute to the molecular specification of the vast complexity of neurons in the cerebral cortex [START_REF] Krishna | Cadherin expression in the somatosensory cortex: evidence for a combinatorial molecular code at the single-cell level[END_REF].

PCDH19 (MIM# 300460), located on chromosome Xq22.3, encodes the 1148 amino acid protocadherin 19, and belongs to the 2-subclass of non-clustered protocadherins that also includes PCDH8, PCDH10, PCDH17 and PCDH18 [START_REF] Wolverton | Identification and characterization of three members of a novel subclass of protocadherins[END_REF]Redies et al., 2005]. The PCDH19 gene has six coding exons, the first exon being unusually large and encoding the entire extracellular domain, composed of six EC domains (Fig. 1, based on the NM_001184880.1 reference transcript corresponding to the longest isoform). PCDH19 expression is spatially and temporally regulated in the central nervous system during mammalian development and shows a unique expression profile among protocadherins. In particular, protocadherin 19 is highly expressed in the developing brain, including the subventricular zone, the intermediate zone, the subplate, specific layers (layers II, IV, V and VI) of the cerebral cortex, the hippocampus and the subiculum [START_REF] Vanhalst | delta-Protocadherins: a gene family expressed differentially in the mouse brain[END_REF][START_REF] Gaitan | Expression of the delta-protocadherin gene Pcdh19 in the developing mouse embryo[END_REF][START_REF] Kim | Spatiotemporal expression pattern of non-clustered protocadherin family members in the developing rat brain[END_REF][START_REF] Krishna | Layer-specific expression of multiple cadherins in the developing visual cortex (V1) of the ferret[END_REF][START_REF] Kim | The expression of non-clustered protocadherins in adult rat hippocampal formation and the connecting brain regions[END_REF][START_REF] Hertel | Absence of layer-specific cadherin expression profiles in the neocortex of the reeler mutant mouse[END_REF][START_REF] Krishna | Cadherin expression in the somatosensory cortex: evidence for a combinatorial molecular code at the single-cell level[END_REF]. Two other isoforms (NM_020766.1 and NM_001105243.1) resulting from the alternative splicing of exon 2 and the existence of two possible acceptor sites for intron 4 (adding a residue at the beginning of exon 5) exist in databases but their existence, distribution and respective roles need to be confirmed and studied at the molecular level.

The precise function of protocadherin 19 remains currently unknown. Yet, other protocadherins were reported to mediate calcium-dependent cell-cell adhesion in vitro and cell sorting in vivo [START_REF] Redies | delta-Protocadherins: unique structures and functions[END_REF] and could regulate the establishment of neuronal connections during brain development and/or remodeling of selective synaptic connections during the early postnatal stage [START_REF] Kim | Spatiotemporal expression pattern of non-clustered protocadherin family members in the developing rat brain[END_REF]. However, in contrast to cadherins, which associate through strong homophilic interactions, the extracellular domain of PCDH19 exhibits specific but weak homophilic adhesive properties, suggesting that the involvement of PCDH19 in cell adhesion could involve heterophilic interactions [START_REF] Biswas | Protocadherin-19 and N-cadherin interact to control cell movements during anterior neurulation[END_REF][START_REF] Tai | Adhesion properties and retinofugal expression of chicken protocadherin-19[END_REF].

Variants Pathogenic Mutations

More than 60 different mutations in PCDH19 have been identified (Table 1A and Fig. 1). All types of DNA alterations are observed, including nonsense mutations, small nucleotide deletions and insertions, mutations altering the splice sites, missense mutations, and intragenic or whole gene deletions. Half of the pathologic allelic variants result in the appearance of premature termination codons, either due to nonsense mutations (18.3%; 17/93 patients, based on review of the literature and personal data), small deletions or insertions leading to a frameshift (25.8%, 24/93) or splice site mutations (3.2%, 3/93). These mutation types are scattered along the PCDH19 gene with the exception of exon 2, in which no variants or mutations have been reported so far. Whole gene or intragenic deletions have been reported in only 6 patients (6.5%, 6/93) [START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF][START_REF] Depienne | Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females[END_REF][START_REF] Vincent | Identification of genomic deletions spanning the PCDH19 gene in two unrelated girls with intellectual disability and seizures[END_REF] but this mutation class has likely been underestimated since most studies used only direct sequencing, which misses heterozygous deletions, to screen PCDH19 (Table 1C). Missense substitutions, which represent 46.2% (43/93) of the mutations are, contrary to other mutation types, all clustered in exon 1, where they affect highly conserved amino acids in the large extracellular domain of protocadherin 19. Several recurrent mutations have been reported in patients, the most frequent being the Asn340Ser missense mutation (Table 1A).

Mutations have been reported in the Leiden Open Variation Database (X chromosome gene database, PCDH19: http://www.lovd.nl/PCDH19).

Polymorphisms and variants of unknown significance

The PCDH19 gene contains few known polymorphisms (Table 2). The most frequent natural variations reported correspond to synonymous substitutions located in exon 1. Rarer, nonpathogenic missense polymorphisms, all clustered in the intracellular domain of protocadherin 19 (exon 6), were occasionally found in patients and in ethnically matched control populations, suggesting that this domain, contrary to the extracellular domain, can tolerate some missense changes. One missense variant located in exon 3 has been identified in a patient with mental retardation but the pathogenic effect of this variant remains uncertain (Table 1B) [START_REF] Tarpey | A systematic, large-scale resequencing screen of Xchromosome coding exons in mental retardation[END_REF].

Biological Relevance

Mutations in PCDH19: a Loss of Function at the Cellular Level

The mutation spectrum in PCDH19 is compatible with a loss of function of the mutated allele.

Messenger RNAs with mutations introducing premature termination codons (PTC) have indeed been shown to be degraded via the nonsense-mediated mRNA decay (NMD) surveillance system of the cell in patients' fibroblasts [START_REF] Dibbens | X-linked protocadherin 19 mutations cause female-limited epilepsy and cognitive impairment[END_REF]. The identification of whole gene deletions in three patients [START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF][START_REF] Depienne | Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females[END_REF]] also supports the notion that loss of function is the main consequence of the mutations. Missense mutations located in the extracellular domain could alter the adhesive properties of protocadherin 19 and also lead to a loss of function. In particular, the Asn340Ser and Glu414Gln mutations could specifically alter amino acids involved in calcium binding [START_REF] Patel | Type II cadherin ectodomain structures: implications for classical cadherin specificity[END_REF][START_REF] Marini | Protocadherin 19 mutations in girls with infantile-onset epilepsy[END_REF]. However, these

hypotheses have yet to be formally tested.

Cellular Interference: a Gain of Function at the Tissue Level

Males with hemizygous PCDH19 mutations show normal cognitive function and do not have seizures, although a subtle psychiatric phenotype was evoked in some asymptomatic carriers [START_REF] Dibbens | X-linked protocadherin 19 mutations cause female-limited epilepsy and cognitive impairment[END_REF]. The absence of major symptoms in hemizygous males indicates that the constitutive loss of function of protocadherin 19 (i.e., the absence of functional protein in all the cells of an individual's body) is not pathogenic. Hence, although protocadherin 19 could be essential for early brain morphogenesis in other species [START_REF] Emond | Protocadherin-19 is essential for early steps in brain morphogenesis[END_REF], it is a non essential protein in humans, its absence likely being compensated for or buffered by other proteins and pathways.

In contrast, females with heterozygous PCDH19 mutations present with early intractable seizures and a variable degree of mental retardation. PCDH19 is located in a region submitted to Xinactivation in females [START_REF] Dibbens | X-linked protocadherin 19 mutations cause female-limited epilepsy and cognitive impairment[END_REF]. Random X-inactivation in mutated females is expected to lead to tissue mosaicism; i.e., co-existence of cells that have inactivated the mutated PCDH19 allele and express the normal protein, and PCDH19-negative cells that have inactivated the normal allele [START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF]. This mosaicism could account for the pathogenesis by altering cell-cell interactions [START_REF] Ryan | Epilepsy and mental retardation limited to females: an X-linked dominant disorder with male sparing[END_REF][START_REF] Dibbens | X-linked protocadherin 19 mutations cause female-limited epilepsy and cognitive impairment[END_REF][START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF]. The loss of function of protocadherin 19 at the level of the cell would thus result in a gain of function at the tissue level because of abnormal interactions between "mutated" and "normal" cells. A mechanism of this type was termed "cellular interference" [START_REF] Wieland | Mutations of the ephrin-B1 gene cause craniofrontonasal syndrome[END_REF] in reference to the "metabolic interference" concept developed many years ago by William Johnson [START_REF] Johnson | Metabolic interference and the + -heterozygote. a hypothetical form of simple inheritance which is neither dominant nor recessive[END_REF]. According to this theory, mosaic males would be affected like mutated females. The identification of an affected male with a mosaic PCDH19 deletion in his fibroblasts, strongly supports cellular interference as the pathogenic mechanism associated with PCDH19 mutations [START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF]. To definitely establish that cellular interference is the pathogenic mechanism, it would however be necessary to demonstrate that females homozygous for PCDH19 mutations are also unaffected like hemizygous males. In the absence of human cases, the development of a PCDH19-deficient mouse model will be crucial to confirm this pathogenic mechanism, assuming that the pathogenic mechanisms are identical in both species.

Interestingly, another human disorder, craniofrontonasal syndrome (CFNS; MIM# 304110), caused by mutations in EFNB1, the gene encoding Ephrin B1, a ligand for Eph receptors (EphR) on chromosome Xq12, has the same unusual X-linked pattern of inheritance [START_REF] Wieland | Mutations of the ephrin-B1 gene cause craniofrontonasal syndrome[END_REF]. Ephrin B1/EphR signaling plays a role in cell migration and pattern formation during developmental morphogenesis [START_REF] Klein | Eph/ephrin signaling in morphogenesis, neural development and plasticity[END_REF], reminiscent of the possible function of protocadherin 19 in brain development. Cellular interference has been confirmed as the pathogenic mechanism in CFNS. Female mice heterozygous for mutations in Ephrin B1 have a mosaic expression of Ephrin B1 resulting in ectopic interactions between the Ephrin B1 ligand and EphB receptors that are sufficient to induce the skeletal defects [START_REF] Compagni | Control of skeletal patterning by ephrinB1-EphB interactions[END_REF].

Clinical Relevance Clinical Features of Patients with PCDH19 Mutations

Female patients with heterozygous PCDH19 mutations have epileptic phenotypes ranging from mild to severe in terms of seizure type and severity. Seizures usually begin in infancy or early childhood (mean age at onset: 12.9 months, median: 10 months; range: 4-60 months; n = 86 patients from the literature and unpublished personal data) and are highly sensitive to fever.

Febrile seizures (FS) are the initial manifestation in approximately half of the cases and seizures are triggered or worsened by fever in 90% of the patients [START_REF] Scheffer | Epilepsy and mental retardation limited to females: an under-recognized disorder[END_REF][START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF][START_REF] Marini | Protocadherin 19 mutations in girls with infantile-onset epilepsy[END_REF][START_REF] Depienne | Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females[END_REF]. Seizure types mostly consist in generalized tonic, clonic or tonic-clonic and/or focal seizures with or without secondary generalization.

Atypical absences, atonic seizures and myoclonic jerks may also be part of the clinical picture, although they are more rarely observed [START_REF] Scheffer | Epilepsy and mental retardation limited to females: an under-recognized disorder[END_REF][START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF][START_REF] Marini | Protocadherin 19 mutations in girls with infantile-onset epilepsy[END_REF][START_REF] Depienne | Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females[END_REF][START_REF] Specchio | Spectrum of phenotypes in female patients with epilepsy due to protocadherin 19 mutations[END_REF]. Status epilepticus, which can be inaugural, and prolonged seizures are frequently reported in PCDH19-positive patients but the most characteristic feature is the presence of brief seizure clusters lasting 1-5 minutes and repeating up to or more than 10 times a day during several days [START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF][START_REF] Marini | Protocadherin 19 mutations in girls with infantile-onset epilepsy[END_REF][START_REF] Depienne | Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females[END_REF][START_REF] Specchio | Spectrum of phenotypes in female patients with epilepsy due to protocadherin 19 mutations[END_REF]. Seizures are resistant to treatment in most cases, especially during infancy and childhood, but their frequency and intractability tend to decrease naturally over time, some patients being sometimes free of seizures during adolescence or adulthood on monotherapy [START_REF] Scheffer | Epilepsy and mental retardation limited to females: an under-recognized disorder[END_REF][START_REF] Depienne | Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females[END_REF][START_REF] Specchio | Spectrum of phenotypes in female patients with epilepsy due to protocadherin 19 mutations[END_REF].

We and others have shown that the clinical spectrum associated with PCDH19 mutations can overlap that of Dravet syndrome (DS, previously named severe myoclonic epilepsy of infancy or SMEI), a stereotyped epileptic encephalopathy also associating FS and epilepsy but due in 75% of cases to a de novo mutation in the SCN1A gene [START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF][START_REF] Marini | Protocadherin 19 mutations in girls with infantile-onset epilepsy[END_REF][START_REF] Depienne | Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females[END_REF][START_REF] Higurashi | PCDH19 mutation in Japanese females with epilepsy[END_REF][START_REF] Nabbout | Protocadherin 19 mutations in girls with infantile-onset epilepsy[END_REF]. However, DS-like patients with PCDH19 mutations slightly differ on average from SCN1A-positive classical DS patients: age at onset is slightly older (12.5 months [range: 4-60] versus 6.3 months [range: 0.5-14]), status epilepticus and occurrence of myoclonic jerks are less frequent, and long-term outcome is better in PCDH19-positive patients than in SCN1A-positive patients, most patients with PCDH19 mutations fitting the definition of bordeline SMEI (SMEB) [START_REF] Fukuma | Mutations of neuronal voltage-gated Na+ channel alpha 1 subunit gene SCN1A in core severe myoclonic epilepsy in infancy (SMEI) and in borderline SMEI (SMEB)[END_REF]. Furthermore, photosensitivity, frequently reported in classical DS, is exceptional in PCDH19-positive patients [START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF][START_REF] Marini | Protocadherin 19 mutations in girls with infantile-onset epilepsy[END_REF].

Behavioral disturbances are frequent in patients with heterozygous PCDH19 mutations and essentially manifest as autistic, obsessive or aggressive features [START_REF] Scheffer | Epilepsy and mental retardation limited to females: an under-recognized disorder[END_REF][START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF][START_REF] Depienne | Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females[END_REF][START_REF] Marini | The genetics of Dravet syndrome[END_REF]. In some patients, social withdrawal or personality disorders are even the most prominent and disabling feature when the patient becomes older [START_REF] Depienne | Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females[END_REF].

Intellectual outcome ranges from normal intellect (27.7%, 23/83), to mild (36.1%, 30/83), moderate (21.7%, 18/83), or severe (14.5%, 12/83) cognitive impairment [START_REF] Scheffer | Epilepsy and mental retardation limited to females: an under-recognized disorder[END_REF][START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF][START_REF] Depienne | Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females[END_REF][START_REF] Marini | The genetics of Dravet syndrome[END_REF][START_REF] Specchio | Spectrum of phenotypes in female patients with epilepsy due to protocadherin 19 mutations[END_REF].

Interestingly, the cognitive prognosis does not appear to be clearly related to the severity of epilepsy [START_REF] Depienne | Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females[END_REF][START_REF] Specchio | Spectrum of phenotypes in female patients with epilepsy due to protocadherin 19 mutations[END_REF]. Language delay is frequently associated with cognitive deficit.

Finally, neurological features such as ataxia can also be observed in some patients and are reminiscent of those observed in DS patients.

Sporadic Versus Familial Cases

Although PCDH19 was first identified in large families where the mutations were inherited over several generations, PCDH19-related epileptic encephalopathy is more commonly sporadic or encountered in families with few affected females, making the recognition of the unusual pattern of inheritance difficult. De novo mutations account for most isolated cases (72%, 32/44) and represent 56% (32/57) of all mutations reported so far, in which transmission could be investigated [START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF][START_REF] Marini | Protocadherin 19 mutations in girls with infantile-onset epilepsy[END_REF][START_REF] Depienne | Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females[END_REF][START_REF] Specchio | Spectrum of phenotypes in female patients with epilepsy due to protocadherin 19 mutations[END_REF]. The remaining mutations found in sporadic cases were inherited by asymptomatic fathers (18%, 8/44), asymptomatic mothers (7%, 3/44) or by a mother who has had only FS (2%). In addition, parental mosaicism leading to the recurrence of the disease was demonstrated in two mothers (one being asymptomatic and one being affected) of unrelated families [START_REF] Dibbens | Recurrence risk of epilepsy and mental retardation in females due to parental mosaicism of PCDH19 mutations[END_REF], a result reminiscent of mosaicism in DS caused by SCN1A mutations [START_REF] Depienne | Mechanisms for variable expressivity of inherited SCN1A mutations causing Dravet syndrome[END_REF].

Genotype-Phenotype Correlations

Since the clinical features (age at onset, severity of the epilepsy and cognitive outcome) are highly variable for a given mutation even within the same family, genotype-phenotype correlation studies will likely be uninformative. Strikingly, Higurashi and collaborators reported monozygotic twin sisters in which the p.Asn340Ser mutation was associated with different clinical pictures, confirming the existence of non-genetic modifiers [START_REF] Higurashi | PCDH19 mutation in Japanese females with epilepsy[END_REF]]. An expected source of phenotypic variability is the status of X inactivation in females. Interestingly, skewing of X chromosome inactivation can occur in normal females and increases in tissues with age [START_REF] Chagnon | Age-associated skewing of X-inactivation ratios of blood cells in normal females: a candidate-gene analysis approach[END_REF][START_REF] Bolduc | No evidence that skewing of X chromosome inactivation patterns is transmitted to offspring in humans[END_REF]. A totally skewed X inactivation situation would theoretically reproduce a non-pathogenic situation. Partially skewed X inactivation would represent intermediate situations in which cellular interference could be limited compared to balanced X inactivation, where it would be expected to be the highest. In this setting, the severity of the epilepsy and/or the intellectual disability could be correlated with the relative amount of inactivated neurons for each chromosome, and the female mutation carriers with a totally skewed pattern of inactivation in the brain would be asymptomatic. However, so far no correlations between the X-inactivation status in blood cells and the phenotypic expression have been found in support of this hypothesis [START_REF] Marini | Protocadherin 19 mutations in girls with infantile-onset epilepsy[END_REF]. Nonetheless, the X-inactivation status in lymphocytes does not reflect that of the neural tissues and studies investigating directly cerebral tissues, in mouse models for example, are important to further investigate this hypothesis.

Diagnostic Relevance

The identification of mutations of the PCDH19 gene provides a definite diagnosis in female patients with infantile epilepsy. This result also makes it possible to calculate the risk of recurrence of the disorder in the family, which is markedly different if the pathogenic mutation is de novo or if it has been inherited from a parent. Although diagnosis is made clinically, differentiating one epileptic condition from another is sometimes difficult, especially when the first symptoms appear. The molecular confirmation of the genetic defect underlying the epilepsy and an analysis of the parents' status are crucial to be able to give families appropriate genetic counseling.

Molecular testing of PCDH19 should be considered in females with early-onset FS and/or epilepsy with or without cognitive impairment and family history. Some clinical features could help to prioritize the analysis of PCDH19, such as female patients presenting with seizure clusters, the presence of multiple affected females in the family with obligate male carriers, and, more generally, the presence of generalized and/or focal seizures beginning in infancy or early childhood, resistant to treatment and sensitive to fever. With regard to Dravet syndrome, screening for PCDH19 mutations should be performed for female patients when analysis of SCN1A is negative. Molecular testing of PCDH19 should include sequencing of the coding sequence of the gene as well as a method (quantitative PCR, multiplex ligation-dependent probe amplification or equivalent) able to identify heterozygous deletions. The percentage of PCDH19positive cases in females with FS and epilepsy has been shown to range from 5 to 37% depending on the clinical criteria [START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF][START_REF] Marini | Protocadherin 19 mutations in girls with infantile-onset epilepsy[END_REF][START_REF] Depienne | Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females[END_REF]. Molecular testing of PCDH19 in sporadic affected males can be considered with the same indication, although the somatic mosaicism expected in this case can easily be missed if the analysis is performed from genomic DNA extracted from blood cells, which considerably complicates the interpretation and decreases the reliability and significance of the result.

Given the unusual mode of inheritance and the wide phenotypic variability associated with PCDH19 mutations, genetic counseling appears delicate. In the case of mutations inherited from an asymptomatic father, all the daughters are expected to be affected. Considering the frequent poor outcome (mild to severe cognitive impairment in about 70% of PCDH19-positive females) of PCDH19-related epileptic encephalopathy, it is feasible to offer a prenatal diagnosis that could simply be based on fetal sex determination from maternal blood [START_REF] Wright | The use of cell-free fetal nucleic acids in maternal blood for noninvasive prenatal diagnosis[END_REF]. Female patients with PCDH19 mutations have a 50% risk of transmitting the mutation but, as only the females would be affected, the overall risk would be 25%. In cases with de novo mutations, the risk of recurrence is expected to be low but the possibility of germinal mosaicism in one parent is still possible [START_REF] Dibbens | Recurrence risk of epilepsy and mental retardation in females due to parental mosaicism of PCDH19 mutations[END_REF].

Future Prospects

Although the frequency and clinical features of PCDH19-related epileptic encephalopathies have become more precisely determined during the past 3 years, several challenges remain to fully understand the functional consequences of the mutations and the mechanisms by which they contribute to epileptogenesis and cognitive impairment.

Several steps were recently made towards elucidating the function of protocadherin 19 in zebrafish or chicken models. The zebrafish ortholog of protocadherin 19 (pcdh19) was shown to be crucial for early steps of brain morphogenesis. Partial depletion of the protein with morpholino oligonucleotides impairs the convergence cell movements of the anterior neural plate, where pcdh19 is specifically expressed, a phenotype reminiscent of the n-cadherin (ncad) mutants [START_REF] Emond | Protocadherin-19 is essential for early steps in brain morphogenesis[END_REF]. Interestingly, the Pcdh19 and Ncad proteins directly interact in vitro and in vivo in this model [START_REF] Biswas | Protocadherin-19 and N-cadherin interact to control cell movements during anterior neurulation[END_REF]. Together with the observation that Pcdh19 exhibits weak homophilic adhesive properties [START_REF] Biswas | Protocadherin-19 and N-cadherin interact to control cell movements during anterior neurulation[END_REF][START_REF] Tai | Adhesion properties and retinofugal expression of chicken protocadherin-19[END_REF], these results suggest that protocadherin 19 could preferentially interact with other members of the cadherin superfamily to regulate cell adhesion, neuronal migration or synapse formation. The development of mouse models is an important step to confirm these hypotheses as well as the cellular interference theory, and to investigate the genetic or epigenetic factors underlying the phenotypic variability observed in the human disorder.

So far, although mammalian genomes contain over 70 protocadherin genes, only two, PCDH19 and PCDH15 (causing autosomal recessive Usher syndrome) have been related to a human Mendelian disorder. Recent studies have suggested that defects in the expression or function of some other protocadherins may be related to neurodevelopmental disorders such as autism, schizophrenia and mental retardation [START_REF] Bray | Screening the human protocadherin 8 (PCDH8) gene in schizophrenia[END_REF][START_REF] Dean | Gene expression profiling in Brodmann's area 46 from subjects with schizophrenia[END_REF][START_REF] Morrow | Identifying autism loci and genes by tracing recent shared ancestry[END_REF].

These findings suggest that protocadherins are likely to play many roles that have yet to be discovered in human disorders. Mutation nomenclature is based on the PCDH19 cDNA reference sequence (NM_001184880.1). Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence.

Figure 1 .

 1 Figure 1. Schematic representation of the mutations, rearrangements and polymorphisms

Acknowledgments and fundings

We thank the families for their participation, the clinicians who referred their patients to our laboratory, and the DNA and cell bank for DNA extraction and cell culture of research samples.

The authors and their projects have been financially supported by GIS Maladies rares, Assistance Publique des hôpitaux de Paris (AP-HP) including the Programme Hospitalier de Recherche Clinique (PHRC) n°P020910/AOR02085, Institut national de la santé et de la recherche médicale (INSERM), and Université Pierre et Marie-Curie (UPMC). The authors declare that they have no conflicts of interest and no financial interest in this study.

Mutation nomenclature is based on the PCDH19 cDNA reference sequence (NM_001184880.1). Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence.


Bénédicte Coulm

Joel Coste

MD Véronique Tardy

MD Emmanuel Ecosse

Michel Roussey

MD Yves Morel

Prof Jean-Claude Carel
email: jean-claude.carel@inserm.fr

Joel Midwife

Coste

Efficiency of Neonatal Screening for Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency in Children Born in Mainland France Between 1996 and 2003

published or not. The documents may come

Introduction

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders of adrenal steroid biosynthesis [START_REF] White | Congenital adrenal hyperplasia due to 21-hydroxylase deficiency[END_REF][START_REF] Merke | Congenital adrenal hyperplasia[END_REF][START_REF] Speiser | Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: an Endocrine Society clinical practice guideline[END_REF] . The commonest form (≈95%) is due to 21-hydroxylase deficiency (21OHD). It affects about one child in 15,000 and results in clinical symptoms that vary with the severity of the enzymatic defect. Classical forms include salt-wasting forms (SW), for which there is a high risk of life-threatening adrenal insufficiency during the first month of life, and simple virilising forms (SV). In both cases, female neonates present with markedly virilised external genitalia.

Non-classical forms can manifest with hyperandrogenism later in life and do not warrant early recognition through neonatal screening. 3β-hydroxysteroid dehydrogenase deficiency (3ß-HSD) is a rare form of CAH that results in the undervirilisation of external genitalia and adrenal insufficiency; it can be detected by 21OHD screening [START_REF] Johannsen | Delayed diagnosis of congenital adrenal hyperplasia with salt wasting due to type II 3beta-hydroxysteroid dehydrogenase deficiency[END_REF] .

21OHD screening is carried out to prevent neonatal death from acute adrenal insufficiency, inaccurate sex assignment in females with complete virilisation and irreversible childhood hyperandrogenism, which may result from incorrect or late diagnosis [START_REF] Speiser | Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: an Endocrine Society clinical practice guideline[END_REF][START_REF] Pang | Worldwide experience in newborn screening for classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency[END_REF] . 21OHD fulfils the usual criteria [START_REF] Wilson | The evaluation of the worth of early disease detection[END_REF] for neonatal screening, with its low cost and the availability of a widely applicable test (17-hydroxyprogesterone, 17-OHP, determination) and has been implemented in many Western countries including the USA and some European countries 3, 7- 9 . However, it remains controversial with three main arguments against routine screening: i) the test has a low positive predictive value, with frequent false-positive results in preterm neonates due to cross-reactions with steroids other than 17-OHP [START_REF] Janzen | Newborn screening for congenital adrenal hyperplasia: additional steroid profile using liquid chromatography-tandem mass spectrometry[END_REF] , ii) the proportion of cases for which screening really contributes to diagnosis is unclear, as most female cases are easy to detect clinically and salt-wasting is often detected before the screening results are obtained and iii) there is a lack of consensus concerning the 17-OHP threshold to be used, due to changes in 17-OHP distribution with gestational age at birth. screening programme in 1996, after a short pilot feasibility study [START_REF] Cartigny-Maciejewski | Neonatal screening of congenital adrenal hyperplasia due to 21-hydroxylase deficiency: Lille experience 1980-1996[END_REF] . However, as in many other countries, routine 21OHD screening was never evaluated. The main objective of this study was to evaluate the efficiency of the national French screening program for 21OHD.

We retrospectively collected real-life screening data and clinical data for affected neonates, to determine whether screening by the Association Française pour le Dépistage et la Prévention des Handicaps de l'Enfant (AFDPHE), a national organisation, had facilitated the identification of cases before clinical diagnosis.

Methods

Population studied and data collected

We carried out a retrospective study on all children born in mainland France between January 1 st , 1996 and December 31 st , 2003. Screening was carried out at 21 regional centres, under the auspices of the Association Française pour le Dépistage et la Prévention des Handicaps de l'Enfant (AFDPHE) [START_REF] Dhondt | The wonderful history of neonatal screening[END_REF] . Blood was collected from three-day-old infants on filter paper and 17-OHP concentration was determined by automated time-resolved fluoroimmunoassay (DELFIA®) or RIA. Infants with 17OHP levels above the threshold applied for screening purposes were evaluated further for the diagnosis of 21OHD. We collected data from the regional centres on all newborns for whom 21OHD screening results

were considered positive.

Statistical analysis

We calculated the sensitivity, specificity and predictive values of the screening test, with 95% confidence intervals (CI) for preterm neonates born before 37 weeks of gestational age, for term newborns, and for both considered together. We classified the contribution of CAH screening to the diagnosis of true positives as follows: screening was considered useful if it led to the diagnosis of classical 21OHD (SW or SV forms) or 3ß-HSD deficiency that was not suspected clinically because there were no symptoms or because the symptoms and signs (genital abnormalities, dehydration) had not been recognised; screening was considered not useful if CAH was diagnosed before the results of screening became available (on the basis of family history, prenatal diagnosis or neonatal systematic examination). Screening was also considered not useful for false negative cases of classical CAH and for children with positive screening results diagnosed with non-classical forms of CAH.

The relationship between gestational age at birth and 17-OHP concentration was studied by linear regression analysis in a sample of 10,523 preterm neonates born before 37 weeks of amenorrhoea selected from the infants testing positive. The values for 17-OHP concentration were not normally distributed and a natural logarithm transformation was therefore applied. Goodness of fit (R 2 ) was calculated for various linear regression models, to identify the factor best predicting 17-OHP concentration: gestational age or birth weight.

Linear regression models were constructed for the imputation of missing data for term or birth weight.

Mortality rates for children under the age of one year were calculated between 1979 and 2007, from CepiDc data. Changes in mortality rate over time were assessed by Poisson regression analysis. We looked for a possible change in slope after 1996 (the year in which the screening programme was generalised), by looking for an interaction between "year", considered as a continuous variable, and "before/after screening introduction" considered as a dichotomous variable.

All analyses were performed with SAS 9.2 software (SAS Institute, Cary, NC, USA).

The study was approved by the CCTIRS and CNIL and was conducted in accordance with French legislation.

Results

During the eight-year study period, 6,012,798 screening tests for 21OHD were performed on children born in mainland France (Figure 1, Table 1 and supplementary Table 1). The laboratory methods for 17-OHP determinations and their thresholds are shown in supplementary We identified 25 children as false negative for 21OHD screening: 23 were reported by genotyping laboratories, and 20 of these cases were also reported by the screening centres, with two reported by the CépiDC. Most of the false negatives (16/25) had SV forms (Figure 1).

Altogether, the incidence of classical 21OHD (SW and SV forms) and 3ß-HSD deficiency in France between 0 and 1 year was 0.78/10 5 births/year, with a 95% CI of [0.70 -0.86] and the prevalence was 1/15699 births (95%CI, 1/17445 -1/14269) (including false negative subjects in their birth cohort). The sensitivity of screening was 93.5% with an overall positive predictive value of 2.3% (Table 3). Sensitivity was higher for SW 21OHD (96.9%, 95%CI, 94.8-98.9) than for SV 21OHD (82.8%, 95%CI, 75.1-90.5). Most falsepositive screening test results were obtained for preterm newborns, for which the positive predictive value of screening was only 0.36%, whereas that for term newborns was 30.4%.

We investigated whether adjustment of the 17-OHP threshold would have improved screening efficiency in preterm newborns, by calculating linear regression models of (positive) 17-OHP levels on filter paper. Gestational age accounted for 9.5% and weight accounted for 7.4% of the variance (R 2 ) of 17-OHP concentration. Adding polynomials and assay techniques increased the R 2 to 10%. Figure 2 illustrates the difficulty of establishing threshold values based on gestational age.

As the primary objective of 21OHD screening is to prevent the death of newborns, we analysed 21OHD-related mortality in France from 1979 to 2007, a 29-year period including the year in which 21OHD screening was introduced. Twenty-one children under the age of one year were classified with an underlying cause of death due to adrenogenital disorders, other and unspecified adrenocortical insufficiency (Figure 3). There was a significant (p=0.002) trend towards a decrease in specific mortality rate during this period, with most of this decrease occurring in 1991 to 1995, before the generalisation of screening. Thus, neither screening itself (yes/no) nor the interaction of screening and time was associated with specific mortality rate (p=0.31 and 0.31, respectively).

Discussion

With the inclusion of 6,012,798 newborns screened in mainland France between 1996 and 2003, this study is by far the largest to date to assess neonatal screening for 21OHD with particular emphasis on its contribution to early diagnosis. We found that sensitivity was good (93%), but that the positive predictive value of screening was low (2.3%), although it improved markedly if we considered only term newborns (30.4%). Screening results contributed to diagnosis in 42% of the cases. Moreover, the large number of infants for which no conclusion was drawn raises questions about the practical organisation of 21OHD screening, due to the large number of false positives.

Table 4 summarises published data from previous population studies, making comparisons with our results possible. The positive predictive values reported in these studies were similar in most cases, with the exception of the Swiss study (positive predictive value of 50%), which presented results for a second determination of 17-OHP on filter paper, rather than those for the primary screening, as in most studies. Unlike previous studies, we took gestational age into account, and we found that screening efficiency differed considerably between term and preterm newborns. Among preterm newborns, there were almost 277 false positives for each case of 21OHD discovered, whereas there were only two to three false positives for each case for term newborns. These difficulties arise from the low specificity of immunological assay techniques for determining levels of 17-OHP in preterm newborns due to high plasma concentrations of steroids other than 17-OHP that cross-react in the assays (with sulphated metabolites), generating false-positive results [START_REF] Nordenstrom | Neonatal screening for congenital adrenal hyperplasia: 17-hydroxyprogesterone levels and CYP21 genotypes in preterm infants[END_REF][START_REF] Wong | Identification of the steroids in neonatal plasma that interfere with 17 alpha-hydroxyprogesterone radioimmunoassays[END_REF][START_REF] Al Saedi | Reference ranges for serum cortisol and 17hydroxyprogesterone levels in preterm infants[END_REF] . Some countries have adopted variable threshold values based on gestational age (Table 4), but our study shows that there is a large overlap of 17-OHP levels between affected and unaffected preterm newborns and that increasing the threshold level in this particular population would result in a loss of sensitivity. One possible alternative is the use of tandem mass spectrometry as a second line test to improve the positive predictive value of screening [START_REF] Janzen | Newborn screening for congenital adrenal hyperplasia: additional steroid profile using liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Minutti | Steroid profiling by tandem mass spectrometry improves the positive predictive value of newborn screening for congenital adrenal hyperplasia[END_REF] . These techniques were recently recommended in the Endocrine Society guidelines 3 , but they are costly, not widely available for population screening and require thorough evaluation, including cost-benefit analyses.

Although 21OHD screening correctly identified 93% of cases, its impact on diagnosis was much smaller, as it contributed to early diagnosis in 45 to 50% of the children identified, corresponding to about 20 children per year in France or an incidence of 2.66/10 5 /year. The main reasons for this minor contribution are that girls with classical 21OHD are readily identified during neonatal paediatric examination and CAH is an autosomal recessive disorder, making prenatal or neonatal diagnosis more likely in families with an index case. In addition, in a small proportion of boys (9/153) with SW forms, adrenal crisis occurred before screening results became available and the children were correctly managed based on their clinical presentation.

Screening for 21OHD is designed principally to decrease neonatal disease-specific mortality. A decrease in specific mortality has been observed over the last three decades, but the timing of this decrease suggests that it was due to improvements in paediatric care rather than to the introduction of screening. The probability of death due to neonatal adrenal crisis in the absence of screening is widely debated and has been reported to vary from 0 to 4% of patients with SW 21OHD in populations with high standards of clinical awareness and care for 21OHD [START_REF] Grosse | How many deaths can be prevented by newborn screening for congenital adrenal hyperplasia?[END_REF] . In our study population of 285 children with salt-wasting 21OHD born between 1996 and 2003 (276 true positives and 9 false negatives), using 4% as an estimate suggests that 11.5 neonatal deaths would have been expected in the absence of screening, a figure to compare to 3 deaths observed during the first year of life. In addition to preventing mortality, screening for 21OHD can prevent inaccurate gender assignment and irreversible childhood hyperandrogenism. In our study, inaccurate gender assignment was not made in the 5 fully virilised females (Prader stage V) but screening allowed the identification of 47/77 patients with a SV form confirming the value of screening to detect the 21OHD before the appearance of severe hyperandrogenism.

Our findings also show that the organisation of screening, as currently conducted in France (and possibly elsewhere), was not satisfactory. Screening centres encountered major operational difficulties with follow-up of the large number of positive tests and no conclusion about status was reached in many cases, raising medical, ethical and responsibility issues.

Questions remain concerning the fate of 307 children for whom successive assays remained above the threshold value but for whom no further follow-up data were obtained. Data for weight and gestational age were also frequently missing, although this information makes it easier to interpret the assay results and should therefore be collected.

Our study was subject to several limitations. The apparent lack of impact of screening on mortality is likely due to insufficient statistical power, given the limited numbers in the mortality analysis (21 deaths in 29 years), the unmeasured effect of pilot 21OHD screening programs during the 1990-1995 period and more importantly undiagnosed 21OHD-related deaths in the period before screening as documented in central Europe [START_REF] Kovacs | Lessons from 30 years of clinical diagnosis and treatment of congenital adrenal hyperplasia in five middle European countries[END_REF] . In addition, inclusion in our analysis of misclassified adrenal diseases other than CAH might have obscured the effect of screening on mortality. However, the study had sufficient power (80%)

to detect a decrease in specific mortality of 65% or more after screening implementation. We could not trace with precision the reasons for which false-negative cases were missed (17-OHP concentration below the threshold, positive test not taken into account correctly, other reason). False-negative cases may have been underestimated for several reasons, including lack of ascertainment if cases were detected later in life and not followed by paediatric endocrinologists or subjected to molecular analyses. We were also unable to determine the number of unnecessary visits and laboratory investigations for children eventually found not to have 21OHD, or their impact on the anxiety of the parents. Missing data on gestational age may have resulted in an overestimation of the sensitivity of the screening, particularly in subgroup analysis (preterm and term newborns). One further limitation is that data on neonatal screening from 1996-2003 are only presented in 2011, at a time when they might be considered less timely. This apparent delay results from long and tedious data collection and monitoring and from the need to wait several years in order to be able to identify FN SV forms since the diagnosis is made as late as 5 or 6 years in some cases. Indeed, we searched for FN cases in 2010, at a time when the youngest child in the cohort was older than 6 years, which is an additional strength of our study.

In France, the budget per screened infant (for 21-OHD only) is €1.23 in the absence of follow-up (US$ 1.7), corresponding to approximately €924,500/year. As ≈ 20 cases of 21OHD are identified by screening each year, this budget corresponds to approximately €50,000 (US$ 70,000) per case. In the US, the cost per screened infant without follow-up has been estimated at US$ 2.3 to 6.0 and applying these figures to our data would result in estimates of US$ 95,000 to 245,000 per case [START_REF] Yoo | The cost effectiveness of screening newborns for congenital adrenal hyperplasia[END_REF] . Assumptions concerning the number of potential deaths among these cases and a complete medical economic analysis of indirect costs might allow a full evaluation of the cost per life-year saved [START_REF] Yoo | The cost effectiveness of screening newborns for congenital adrenal hyperplasia[END_REF] .

Overall, we found that neonatal 21OHD screening was efficient in term newborns, with a variable impact on clinical management given that most affected female newborns are easy to identify without screening. By contrast, the efficiency of screening was very low in preterm newborns, resulting in large numbers of false positives, flooding the system and leading to its dysfunction and leading to the identification of 6 cases of potentially lethal saltwasting 21OHD among more than 10,000 positives in 8 years. Improved organisation might have prevented this dysfunction and allowed a comprehensive follow-up of all positive cases.

However, a decrease of the false positive cases is necessary to improve efficiency.

So, what recommendations should be made based on our results? We recommend that newborn screening for 21OHD is continued for term newborns in areas in which it is already performed and that careful consideration is given to its implementation in areas in which this is not the case. By contrast, we recommend that 21OHD screening, as performed in this study, should not be carried out for preterm newborns since the positive predictive value of the test is very low and most preterm newborns are subject to careful paediatric care that should ensure that incipient salt wasting adrenal crises are readily recognised. In France, the national neonatal screening organisation and representatives of professional organisations in neonatology, paediatric endocrinology and rare endocrine diseases are currently discussing how to improve the national screening program. Our improved program, as well as others around the world will have to be carefully evaluated. Population-based studies with a sample size >100 000 were selected

Legends for figures

Figure 1 .Figure 2 . 17 -

 1217 Figure 1. Results of neonatal screening for 21-hydroxylase deficiency in France 1996-2003

Figure 3 .

 3 Figure 3. Specific mortality rates due to adrenogenital disorders, other and unspecified adrenocortical insufficiency during the first year of life in France, 1979-2007.

Table 1 .

 1 Neonatal screening tests were positive for 15,407 newborns, with 370 considered affected, 11,324 considered unaffected and no conclusion reached by the screening centres for 3,132. For 1,814 infants, the conclusion was discordant with the last recorded 17-OHP concentration (n = 338 considered unaffected with a last 17-OHP

determination considered positive and n = 1476 considered unaffected without the recorded monitoring of 17-OHP concentrations). 581 children were identified as deceased : most of these children were preterm and, in all cases, the death of the child was considered by clinical centres to be unrelated to 21OHD (Figure

1

). Most of the newborns with positive results for 21OHD screening were born before term (91% of those for whom data were available). The median day for filter paper sampling was day 4, although the screening protocol called for sampling on day 3. Of the 370 newborns considered to be affected, 358 had a classical form of 21OHD (n = 354) or 3βHSD (n = 4) deficiency and 12 had a non-classical form of CAH.

The median age at diagnosis of CAH was seven days (Table

2

). Weight loss was severe (>10% of expected body weight) in 19% of those for whom data were available, and plasma sodium concentration was below 130 mmol/l in 18% of the infants. Screening was useful for diagnosis in 162 of the 358 children with classical CAH and positive screening results, mostly males with the SW form (n = 106/162). Screening results were positive but not useful for diagnosis in 74 children with a family history of 21OHD and in 96 girls with genital abnormalities detected during neonatal examination. In addition, screening results were positive in 13 boys with classical 21OHD who were diagnosed clinically before the screening results became available. Of interest, among the 38 premature babies with positive screens, screening was useful to the diagnosis in only 13 among whom only 6 had a SW form.

Table 1 -Principal characteristics of newborns with positive screening results for CAH in mainland France between 1996 and 2003 N = 15,407

 1 imputations of gestational age from sex and birth weight: n = 998, imputations of birth weight from sex and gestational age: n = 2653; † missing data: sex n = 158; age at screening n = 1117; 17-OHP screening result n = 72; 17-OHP measurement method n = 5.

	Sex
	Male

Data are presented as numbers (percentages) unless otherwise stated; †, median, interquartile range; WA: weeks of amenorrhoea; *

Table 3 . Efficiency of 21OHD screening as a function of gestational age at birthTable 3a :

 33a Raw data

					Screening	Affected	Unaffected	Total
					Positive	358	15,049	15,407
	All	newborns	Negative Total	25 383	5,997,366 6,012,415	5,997,391 6,012,798
					Positive	318	740	1058
	Term	newborns	(≥37 WA)	Negative Total	21 339	5,578,196 5,578,936	5,578,217 5,579,275
					Positive	38	10,524	10,562
	Preterm	newborns	(<37 WA)	Negative Total	2 40	422,959 433,383	422,961 433,523

Table 3b :

 3b Efficiency calculationsData are expressed in % [95% confidence interval]; efficiency in term and preterm newborns was calculated for those without missing data for gestational age at birth(11,620/15,407).

	All	newborns	Positive predictive value Negative predictive value	2.3 [2.1-2.6] 99.9 [99.9 -99.9]	Specificity Sensitivity	99.7 [99.7 -99.7] 93.5 [90.9 -95.9]
	Term	newborns	(≥37 WA)	Positive predictive value Negative predictive value	30.1 [27.3-32.8] 99.9 [99.9 ; 99.9]	Specificity Sensitivity	99.9 [99.9-99.9] 93.8 [91.2-96.4]
	Preterm	newborns	(<37 WA)	Positive predictive value Negative predictive value	0.4 [0.2-0.5] 99.9 [99.9 ; 99.9]	Specificity Sensitivity	97.6 [99.5-97.6] 95 [83.1 -99.4]

Table 4 . Efficiency of 21OHD screening in published studies

 4

	Reference 22	Country USA (Texas)	Number of newborns 1,936,998	17-OHP threshold (nmol/l) 123	Variable 17-OHP threshold with term yes	Sensitivity (%) 86	Positive predictive value (%) NA
	11	France	408,138	36 to 60	no	89	2.1
	23	Switzerland	333,221	30 to 90	yes	97	50.0
	24	Netherlands	176,684	60	yes	100	5.9
	25	Italy	128,330	36	yes	NA	1.9
	This study	France	6,012,798	40 to 100	no	93	2.3

Acknowledgment

The authors wish to acknowledge the role of Unité de Recherche Clinique Paris-Cochin and in particular Christelle Auger and Sandra Colas. Cécile Klochendler, Sandrine Borderie and Pr Jean-Marc Treluyer from Unité de Recherche Clinique Paris-Cochin are also acknowledged. Louise Montagne, Claire Jeandel, Kasia Marchal, Candace Bensignor, Chaza Fallouh and the Centres of Clinical Investigation of Lyon and Bordeaux also participated in data collection.

Authors' contributions statement

Jean

Source of funding statement

The study was funded by the French Ministry of Health "PHRC, Programme

Conflict of interest statement

No conflict of interest to disclose.