Ph.D Lotte Munch Lambertsen

Ph.D Lotte Munch Lambertsen

Ph.D.; Kim Ekelund

Ian C Skovsted

Anders Liboriussen

Ph.D Hans-Christian Slotved

H-C Slotved

Characterisation of invasive group B streptococci from adults in Denmark 1999 to 2004

Keywords: Streptococcus agalactiae, protein typing, R4, Cα, Cβ, CPS serotyping Streptococcus agalactiae, protein typing R4, Cα, Cβ, CPS serotyping. 18 19 20 21 22

The aim of this study was to characterise the GBS isolates causing severe invasive infections in patients (>15 years) in Denmark from 1999 to 2004. Methods: 411 invasive GBS isolates were phenotypically characterised by CPS serotype and protein Cα, Cβ and R4. Results: The incidence of invasive GBS disease ranged from 2.2 to 3.2 per 100,000 adults in the study period, being highest among adults above 65 years of age. Diabetes was observed in 15% of the cases, 12% had alcohol abuse and 7% had cancer. Of all isolates 77% were CPS serotypes Ia, Ib, III or V. The surface proteins Cα or R4 were detected as the only protein in 57% of the GBS isolates. Cβ was detected in 12% of the isolates, but always in combination with either Cα or both Cα and R4. Conclusions: The incidence of invasive GBS infections continued to increase in Denmark from 1999 to 2004. In that period, the overall case fatality was 14%. The most prevalent CPS serotypes were serotypes III, Ia, V and Ib. The most prevalent surface protein was R4 when testing for R4, Cα, Cβ. There was no clear relation between the GBS phenotype and infections with fatal outcome.

INTRODUCTION 1

Invasive infections among non-pregnant adults caused by Streptococcus agalactiae, also known as 2 Lancefield group B streptococci (GBS), is a well-known phenomenon [1][2]. 3

The reported incidence of invasive GBS infections in adults varies from 3 to more than 20 per 4 100,000 persons, depending on age and health. The incidence is particularly high among the elderly 5 above 65 years of age [3][1][4]. Henning et al [5] also found that there was a higher incidence of 6 invasive disease with GBS infections among elderly residing in nursing homes compared to 7 community residents. In particular elderly persons aged 65 years or more with an underlying 8 medical disease seem to be at a higher risk of contracting invasive GBS infections than other adults. 9

The underlying medical conditions and/or chronic diseases which seem to be most important in 10 relation to invasive GBS infections were diabetes mellitus, neurologic diseases, cirrhosis or other 11 liver diseases, stroke, breast cancer and renal failure [6][7][1][4]. In summary, adults at risk of 12 getting a severe GBS infection seem to be adults with reduced immunesystems [4]. 13 The case fatality rate of invasive GBS infections among adults varies from 5 to more than 15% 14 depending on age [3][4] [7]. Blancas et al [7] reported that 9.3% of patients admitted to a hospital in 15 Barcelona, Spain, with a positive GBS blood culture died; it was found that the presence of shock 16 and cancer were factors associated with risk of dying from the disease [7]. In 2005, Edwards and 17 Bakers [8] reviewed the situation in the United States and found the case fatality rate among elderly 18 to be estimated to 14.5%. Per 100,000 persons, this case fatality rate was higher than the estimated 19 case fatality rate due to group A streptococcal infections but lower than the estimate for 20 pneumococcal infections [8]. 21

In Denmark, a study on invasive infections caused by haemolytic streptococci in the period 1999 to 22 2002 showed that invasive infections due to GBS among non-pregnant adults had increased slightly 23 during the last years, and that the incidence of invasive GBS infections was higher for elderly above 65 years than for younger adults. The case fatality rate was 19% [6]. With a high incidence in 1 certain age groups and a relative high case fatality rate, invasive GBS infections are of high 2 importance, and GBS is a bacterium which should be surveyed carefully. 3 4 GBS isolates can be phenotypically characterised based on surface components such as capsular 5 polysaccharides (CPS) and surface proteins. Based on structural differences in polysaccharide 6 capsules, GBS isolates can be divided into ten different serotypes: Ia, Ib, and II to IX [9]. A CPS 7 serotyping method based on a precipitation reaction test, using serotype specific antisera against the 8 capsules and hot acid extracts of the bacteria, has been used in many laboratories since it was 9 introduced by Dr Lancefield in the 1930s. The phenotypic CPS serotype can also be determined 10 using other methods such as GBS latex, ELISA or ouchterlony immuno-diffusion test [10][9]. Some 11 isolates are not typeable using these methods and are, therefore, assigned NT. Another phenotypic 12 characterisation method is to test for the presence of different GBS surface proteins [11][12]. 13 Proteins, which in other studies have proven useful markers to test for are Cα, Cβ, R, and Alp 14 proteins [12][13][14]. Capsular polysaccharides as well as surface proteins are useful typing targets 15 as they are the components recognised by the immune system and, therefore, also possible 16 components of future vaccines [12][15]. 17

The aim of this study was to characterise GBS isolates causing severe invasive infections in patients 18 (>15 years) in Denmark from 1999 to 2004. Phenotypic characterisation methods were used to 19 evaluate the CPS serotypes and protein profile distributions among the GBS isolates as well as to 20 relate these results to infected patients, their age, sex and outcome of infection. 21

22

Materials and Methods 23

Clinical isolates and patient data 24

A total of 411 invasive GBS isolates (from blood and cerebrospinal fluid) isolated from 1999 to 1 2004 from patients (> 15 years old) and received from the Danish departments of clinical 2 microbiology laboratories were included in the study. [START_REF] Ekelund | Invasive group A, B, C and G 22 streptococcal infections in Denmark 1999-2002: epidemiological and clinical aspects 23 8[END_REF] [6] estimated for the 3 period of 1999 to 2002 the proportion of all invasive GBS isolates sent in to be 58% percent. Only 4 one isolate per patient was included in this study. All isolates were confirmed to be GBS by 5 inspection of colony morphology on 5% blood agar plates (SSI Diagnostica, Statens Serum Institut, 6 Hillerød, Denmark) as well as with a group specific agglutination test using group B latex serum 7 (Oxoid A/S, Greve, Denmark). All isolates were stored at -80C in nutrient beef broth containing 8 10% glycerol (SSI Diagnostica). 9

All patient data used in this study was approved by the Scientific-Ethical Committees for 10

Copenhagen and Frederiksberg (no. (KF) 11-156 ⁄ 00) and by the Danish Data Protection Agency 11 (no. 2001-41-0807). Patient data was obtained using a questionnaire filled in by the clinician. 12 13

CPS serotyping 14

The majority of the isolates were serotyped with GBS latex agglutination test (SSI Diagnostica), 15 and all isolates were serotyped/confirmed using the capillary precipitation method (Lancefield 16 method) [16][10]. The capillary precipitation method was performed using serotype specific GBS 17 antisera (Ia -IX) (SSI Diagnostica) and capsular antigens extracted with both 0.1 and 0.2 N HCl 18 (Lancefield extraction method). The latex agglutination test was performed as recommended by the 19 manufacturer (SSI Diagnostica). Non-serotypeable isolates were designated NT. 20 21

Surface protein antigen detection (Cα, Cβ and R4) 22

Cα, Cβ and R4 (also named Rib [17]) specific polyclonal rabbit antisera (SSI Diagnostica) were 23 used to detect surface proteins using a western blot procedure previously described by Slotved et al 24 [9]. Briefly, GBS was grown in Todd Hewitt broth overnight at 36ºC and 5% CO 2 . The bacterial 1 culture was mixed with NuPAGE sample buffer (Invitrogen, Carlsbad, CA) and NuPAGE reducing 2 agent (Invitrogen) and heated for 10 min at 70°C. Ten microliters of sample mixture was loaded on 3 a gel (Tris-Bis 4-12% gradient gel, NuPAGE, Invitrogen) and the gel was run for 1 h at 180 V with 4 NuPAGE running buffer (Invitrogen) on the electrophoresis apparatus XCell4 Surelock Midi-Cell 5 (Invitrogen). After electrophoresis, the proteins were transferred to a nitrocellulose membrane 6 (Invitrogen). The membrane was incubated in blocking buffer (50 g skimmed milk powder (Matas 7 A/S, Allerød, Denmark) added to 1L washing buffer (1×PBS with 0.05% Tween 20) (SSI 8 Diagnostica)). Anti Cα, -Cβ or -R4 polyclonal rabbit sera (SSI Diagnostica) were used as primary 9 antibodies (diluted 1:200), and alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G 10 (Sigma Aldrich, St. Louis, MO) was diluted 1:1000 and used as the secondary antibody. 5-bromo-4-11 chloro-3-indolylphosphate nitro blue tetrazolium (Sigma) was used as a substrate for the colour 12 reaction, which was stopped by the addition of MQ water. 13 14 RESULTS

15

Incidence and seasonal variation of invasive GBS 16

During the six years from 1999 to 2004, the incidence per 100,000 adults (calculated on the basis of 17 the number of received isolates per 100,000 persons above 15 years) increased slightly from 1.3 in 18 1999 to 1.7 in 2004 with at maximum of 1.9 in 2002. Based on an estimated proportion of invasive 19 isolates of 58% [6] as described in Materials and Methods, the incidence of invasive GBS infections 20 in Denmark ranged from 2.2 to 3.2 per 100,000 adults in the study period. 21

The seasonal distribution of invasive GBS isolates per year showed large variation with . However, 22 there was a tendency that a higher number of invasive GBS infections were registered in the four 23 months from August to November, compared with the rest of the year (Fig. 1).

Age and sex dependent incidence, case fatality and underlying medical condition 2

The GBS isolates were from patients with a mean age of 62.8 years ranging from 16 to 99 years of 3 age; 53% (n=219) were females. In the study, we received information on 96% of the patients 4 concerning outcome of invasive GBS infection and underlying medical condition. The overall case 5 fatality was estimated to be 14% (n=58). Of these 58 cases with fatal outcome, 51.7% were males. 6

In the age group below 40 years, 50 isolates were obtained from females and 12 isolates were from 7 males i.e. the age dependent incidence of invasive GBS infections was 1.1 for females and 0.2 for 8 males. The case fatality in this age group was 5.2% for females and 7.7% for males (Fig. 21). In 9 contrast, for the age group above 65 years the number of isolates received was 83 isolates from 10 males and 94 isolates from females with a corresponding incidence of invasive GBS infections of 11 5.7 for males and 3.6 for females. The case fatality in this age group was 17% for both males and 12 females (Fig. 21). As to underlying medical conditions, diabetes was observed in 15% of the cases 13 with a GBS infection, 12% had an alcohol abuse and 7% had cancer. Furthermore, 7% had had a 14 recent operation and 7% had recently delivered (data not shown). 15

CPS serotype and protein distribution of all isolates 17

Of all GBS isolates, 77% were tested to be CPS serotype III (n = 121), Ia (81), V (77), or Ib (38) 18 (Fig. 32a). No isolates of CPS serotypes VI or VII were found among the received isolates. 19 However, among the 2004 isolates, one isolate of the recently defined CPS serotype IX was found. 20 A total of 11% of the isolates were NT with no CPS serotype detected (Fig. 32a). 21

The isolates were also tested phenotypically for the presence of the three surface proteins Cα, Cβ, 22 and R4. The surface proteins Cα or R4 were detected as the only protein in 57% of the GBS isolates 23 (Fig. 32b), while Cβ was detected in 12% of the isolates, but always in combination with either Cα 24 or both Cα and R4. In 22% of the isolates, none of the three surface proteins were detected (Fig. 1 32b). 2 Upon combining the CPS serotyping results with the protein typing results, phenotype III-R4 was 3 found to be the most dominant combination with 18% of all isolates. Twelve percent of the isolates 4 were typed to be phenotype Ia-Cα and 11% were phenotype V-R4 (Fig. 3c). Within CPS serotype 5 III isolates, R4 was detected as the only protein in 62% of the isolates; among CPS serotype Ia 6 isolates, Cα was detected as the only protein in 60% of the isolates and among CPS serotype V, 7 58% tested positive for protein R4 (Fig. 32c). 8

During the six years of the study period, CPS serotype III was the dominating type each year except 9 for 2002, when serotype Ia isolates were present in a higher number (data not shown). CPS serotype 10

V was observed to increase in number over the period from 1999 -2004. During the six years, the 11 protein serotype R4 was detected in most isolates per year except for 2002, when protein type Cα 12 was detected in most isolates (data not shown). In the year 2004, protein R4 was detected in a large 13 number of isolates. Upon combining both typing methods, the high number of R4 protein was 14 explained by a high number of isolates with phenotypes III-R4 and V-R4. 15 16 CPS serotype and protein distribution of isolates from cases with fatal outcome 17

Of GBS isolates from cases with a fatal outcome, 69% were tested to be CPS serotype III (n = 16), 18 Ia (12) or V (12) (Fig. 3d) and 7% of the isolates were NT with no CPS serotype detected (Fig. 19 32d). The surface proteins Cα or R4 were detected as the only protein in 53% of the GBS isolates 20 from fatal cases, while Cβ was detected in 12% of the isolates, but always in combination with both 21 Cα and R4 (Fig. 32e). In 24% of the isolates, none of the three surface proteins were detected (Fig. 22 32e). When combining the CPS serotyping results with the protein typing results, 26% of the 23 isolates from fatal cases were tested to have phenotypes III-R4 or V-R4 (Fig. 32f). 24

The case fatality based on the infecting CPS serotype varied from 0% to 28.6%, none of all patients 1 infected with serotype IX had fatal outcome, 8.9% of all patients infected with an NT and 28.6% of 2 all patients infected with CPS serotype VIII (data not shown). In addition, the case fatality seemed 3 to be related to the sex of the infected patient (data not shown); i.e., 83.3% (Nn=12) of all cases 4 with a fatal outcome and infected with a CPS serotype Ia, were women, 62.5% (Nn=16) of all cases 5 with a fatal outcome and infected with a CPS serotype III were women whereas 66.7% (Nn=12) of 6 all cases with a fatal outcome and infected with a CPS serotype V were men (data not shown); 7 however, the number of isolates in each group was low. 8 9 DISCUSSION 10

This study presents data on invasive GBS isolates from patients (>15 years) in the period from 1999 11 to 2004 in Denmark. The overall incidence of invasive GBS infection in adults in the study period 12 was found to increase slightly from 1.3 per 100,000 adults in 1999 to 1.7 in 2004. Based upon this, 13 only approximately 58% of all GBS isolates identified were received [6], the estimated incidence in 14 the period ranged from 2.2 to 3.2 per 100,000 adults. However, the true incidence of GBS was 15 probably higher as this estimate did not take into account cases where viable isolates were not 16 cultured or detected due to for example treatment with antibiotics prior to diagnostic culturing. 17

Comparing these data with similar data from an earlier time period 1988 to 2002 but from the same 18 geographic area presented in the study by Ekelund et al [6], the incidence has slowly increased in 19 Denmark from 1988 with an incidence below 0.5 per 100,000 inhabitants until 2004 with an 20 incidence of 2.9 per 100,000 adults. A similar incidence and increase in the incidence of invasive 21 GBS has been observed in our neighbouring countries. In Sweden, an annual incidence of 2.4 per 22 100,000 inhabitants in the period 1999-2001 was found on invasive GBS infections. However, this 23 includes the age group below 15 years of age [18]. In Norway, an average incidence in the age 24 group above 19 years of age were found to be 1.34 in 1996-1998 rising to 3.1 per 100,000 in 1999-1 2006 [13]. Data on the incidence of invasive GBS infections from other parts of the world show, 2 however, a higher incidence. In a study from USA, it was found that the incidence in the age group 3 above 18 years increased from 3.6 cases per 100,000 persons in 1990 to 7.3 in 2007 [1]. In another 4 study from USA, the overall incidence of invasive GBS in adults was found to be 6.0 per 100,000 5 persons in 1999 and 7.9 in 2005 [4]. In conclusion, the incidence of invasive GBS infections in 6 adults seems to be increasing not only in Denmark but in several countries. 7

Looking at the seasonal variation (Fig. 1), there was a tendency that a higher number of invasive 8 GBS infections was registered in late summer and autumn compared to the rest of the year, thus the 9 number of isolates per month per year was low. This observation is however similar to the 10 observation in the study performed by Phares et al [4], in which they observed a seasonal variability 11 with distinct peaks occurring each year during the later summer months. The reason for this is 12 unclear, but the observation may provide information which may be helpful in finding out why the 13 incidence of invasive GBS infections is increasing. 14 15 Elderly patients (>65 years) was the age group with the highest incidence of invasive GBS 16 infection; as to sex dependent incidence in this age group males had a higher incidence of invasive 17 GBS compared to females (Figure 21). This is consistent with other studies finding that incidence 18 of invasive GBS increases with age [13][4][1] and that a higher proportion of invasive isolates were 19 from males [1]. The most common underlying medical condition of patients with invasive GBS 20 infection observed in this study was diabetes (15%), alcohol abuse (12%) and cancer (7%). Finding 21 that a specific medical condition can be a possible risk factor for obtaining an invasive GBS 22 infection has also been observed in similar but much larger studies. Skoff et al [1] found that 44% 23 of the adults with an invasive GBS infection had diabetes, 15% had cancer and 7% of the cases had an alcohol misuse and Phares et al [4] found that 41% of cases with invasive GBS infection had 1 diabetes and 17% cancer. The case fatality in this study was 14% which is within the rates found in 2

other recent studies [4][3][8]. 3 4
The majority of the 411 GBS isolates found in this study were tested to be serotype III (29.4%), Ia 5 (19.7%), V (18.7%) and Ib (9.2%) of which serotype III was the most prevalent type (Fig. 32a). 6

Comparing our GBS serotype distribution with a study from another Nordic country (Norway), 7 including 465 GBS isolates from persons above 1 year of age in the period of 1996-2005, showed a 8 similar distribution with the majority GBS isolates tested to be serotype III (24.4%), V (24.1%), Ib 9 (15.1%) and Ia (14.6%) [13]. In Sweden, a smaller study testing 111 isolates from adults in 1998-10 2001 found 42% serotype V and 25% III. Looking at studies performed geographically further away 11 from Denmark, Phares et al [4] tested 4987 isolates collected from nonpregnant paediatric and adult 12 cases in the period of 1999-2005 in USA. They found that the majority of the GBS isolates were 13 serotypes V (31%), Ia (24%), II (12%) and III (12%). Skoff et al [1] performed a similar study 14 testing 1933 isolates collected from nonpregnant adult cases in the period of 2005-2006 in USA, 15 and found that the majority isolates were serotypes V (29.2%), Ia (24.3%), II (13.4%) and III 16 (11.4%). Edwards and Baker [8] presented data on 589 invasive isolates from nonpregnant adults 17 from USA in the period 1992-1999, in which the majority of the 589 tested GBS isolates were 18 serotypes V (27.2%), Ia (24.3%), III (16.5%), Ib (12.2) and II (11.9%). In the three studies from 19 USA the most prevalent serotype were found to be serotype V, compared to serotype III in two of 20 three Nordic studies. Finally, a study by Lachenauer et al [19] showed that in Japan serotypes VIII 21 and VI were very dominant, although this was colonizing isolates from pregnant women in Japan. 22

The observed differences in dominating GBS serotypes in different geographic areas around the 23 world and in different time periods show that a possible future effective global GBS vaccine needs 24 to cover a high number of the serotypes, a fact also concluded by Maione et al. [20]. Another 1 solution to limiting the number of included serotypes is to include a relevant protein component in 2 the vaccine. 3

In this study, the prevalence of three proteins (Cα, Cβ and R4) and distribution within each serotype 4 were tested on the invasive GBS isolates (Fig. 3). Our data showed that the R4 protein was the most 5 prevalent among isolates and associated particularly with serotypes III and V whereas Cα was 6 found in isolates with serotype Ia. Cα and Cβ were often found in serotype Ib isolates (Fig. 32). In 7 other studies [13][14], similar patterns and relations between proteins and serotypes have been 8

found. However, as also seen in other studies [13][14], the data presented here showed that it was 9 possible to find the three proteins in isolates of almost all known GBS serotypes (Fig. 32). Looking 10 at the protein data from a vaccine point of view, this study supports the view of for example Persson 11 et al [14] proposing the R4 protein in a conjugated vaccine with polysaccharide from several of the 12 serotypes to be an ideal possible vaccine formulation. 13

Studies like this estimating the prevalence of serotypes and protein profiles of isolates from 14 different parts of the world are needed to estimate the effectiveness of possible future vaccine 15 formulations. A major goal of a possible vaccine would be to prevent invasive GBS infections and 16 thus also fatal outcomes of the infection. It has been speculated that GBS isolates with certain CPS 17 serotypes or proteins profiles would be related to fatal outcome. In this study we found the serotype 18 distribution of isolates from cases with fatal outcome to be approximately the same as for all 19 analysed isolates (Fig. 32d), however the number of isolates belonging to each CPS serotype from 20 fatal cases was too low to make clear conclusions. For the protein serotype distribution of isolates 21 from cases with fatal outcome, no isolates with both Cα and Cβ proteins were found (Fig. 32e). In 22 contrast, a higher proportion of isolates with protein serotypes Cα, Cβ, R4 (12% compared to 6%) 23 and Cα, R4 (10% compared to 6%) from cases with fatal outcome (Fig. 32e) was detected 24 compared to GBS isolates from cases surviving the GBS infection (Fig. 32b). Apart from this, the 1 protein serotype distribution of the isolates from cases with fatal outcome was approximately the 2 same as among isolates from all analysed isolates, however the number of isolates belonging to 3 each protein serotype from fatal cases was too low to make clear conclusions. When combining 4 both CPS and protein typing results, the number of each phenotype was far too low for making clear 5 conclusions (Fig. 32f). In conclusion, taking into account the low number of isolates from fatal 6 cases, there was a tendency that similar patterns with regard to CPS serotype, proteins profile, and 7 sex distribution were observed for isolates from fatal cases and all analysed isolates. This was also 8 observed by Phares et al 2008 [4]. We are aware that to be able to make statistically significant 9 conclusions on the GBS isolate types from fatal cases and from non-fatal cases a higher number of 10 cases would be needed however in a small country like Denmark this can be difficult. Anyhow, we 11 still find it informative to report observations related to fatal out-come and patients clinical 12 conditions if that information is available even though some studies are small. 13 14

The overall conclusions from this study are that in Denmark from 1999 to 2004, the incidence of 15 invasive GBS infections in adults, and in particular in elderly adults continued to increase, and the 16 case fatality was 14%. The most prevalent CPS serotypes were serotypes III, Ia, V and Ib and the 17 most prevalent surface protein was R4 when testing for R4, Cα, Cβ. There was no clear relation 18 between the GBS phenotype and cases with fatal outcome. Based on the comments from the reviewers we have revised our manuscript.

In the revised manuscript the changes are inserted with Microsoft words track changes. These revisions are also specified below.

Reviewers' comments:

Reviewer #1: The seasonal data do not appear to be significant -the authors should not speculate on insignificant data. We agree with reviewer and have therefore deleted figure 1 and only written in the text, that we observed a seasonal tendency (see page 6. line 22-25 and page 10, line 10-12). Because of this figrure numbers for the remaining two figures have been changed thoughout the manuscript.

This is a major problem in the discussion of the mortality data -this database should be used to do a multivariate analysis of the associations -both clinical and serotype -that appear to predict mortality -the authors can then speculate on any significant associations -at the moment the speculations seem to be based on % differences which is inadequate.

The main focus of this study was typing of the invasive GBS isolates from adults in Denmark. Thus as we also had information on fatal or not fatal outcome of the GBS infection and some information on the patients clinical conditions we found it relevant to add this information even though that it is impossible to make statistical significant conclusions as the study is too small for this. However to make it clear for the reader, that the added clinical information is of limit character, we have added several sentences to the manuscript to emphasis this.

We have added the sentence on page 12 line 19-20: "however the number of isolates belonging to each CPS serotype from fatal cases was too low to make clear conclusions" and on page 13, line 2-3: "however the number of isolates belonging to each protein serotype from fatal cases was too low to make clear conclusions".

We have also added a sentence on page 13, line 8-12: "We are aware that to be able to make statistically significant conclusions on the GBS isolate types from fatal cases and non-fatal cases a higher number of cases would be needed however in a small country like Denmark this can be difficult. We find it informative to report observations related to fatal out-come and patients clinical conditions if that information is available even though some studies are small."

The major difference in incidence by sex in younger adults is striking, but here again statistical analysis is needed to see if that difference is real.

We believe that the incidence of females is higher than males in younger adults due to the fact that some of these women have recently delivered, increasing the risk of getting an invasive GBS infection. Unfortunately we did not have proper registration on how many of the women it was.

We hope that our response has made the manuscript acceptable for publication in your journal.

Fig. 1 . 4 Fig. 21 . 7 Fig. 32a )

 1421732a Fig. 1. Average numbers of invasive GBS isolates per month for six years, from 1999 to 2004 in 2

of invasive group B streptococci from adults in Denmark 1999 to 2004".

	Figure 1 Figure 2
							Our ref.
						9 March 201010
		European Journal of Clinical Microbiology &
	2a	Infectious Diseases	2d
			NT				NT
		IX 0,2% VIII 1,7%	10,9%	Ia 19,7%	VIII 3,4%	IX 0,0%	6,9%	Ia 20,7%
		Ib 9,2% II Ref.: MS. No. EJCMID-D-09-00627 V 18,7%	V 20,7%			II Ib 6,9%
		IV Dear Editor,		5,6%	IV			8,6%
		4,4%			5,2%
		III 29,4% Thank you very much for accepting our manuscript for review and for sending re-III 27,6% viewers comments on: "Characterisation
	2b			2e
			not tested				not tested
		tested negative 22%	2%	R4	tested negative 24%	0%	R4
				36%				35%
		Cα,Cβ,R4
		6%			Cα,Cβ,R4
		Cα,R4			12%
		7%
		Cα,Cβ 6%	Cα 21%		Cα,R4 10%	Cα,Cβ 0%	Cα 19%

ACKNOWLEDGEMENTS 22

We thank the Danish departments of clinical microbiology for sending isolates to the National 23 Neisseria and Streptococcus Reference Center. Clinicians are also thanked for using time on filling 24


Sébastien Boisgérault
email: sebastien.boisgerault@mines-paristech.fr

Eric Vecchié
email: eric.vecchie@mines-paristech.fr

MODELLING AND OPTIMIZATION OF DATA-DRIVEN SCENE GRAPHS

Keywords: Modelling, optimization, scene graph, vector graphics, user interfaces, safety critical systems, embedded systems, mobile systems, real-time systems

This article presents data-driven scene graphs, a set of models that address the needs of safety-critical user interfaces design. Data-driven scene graphs merge a description of the user interface behavior as a data-flow program with a description of its graphics content as a hierarchical structure of vector and raster elements. We present a formal description of these models, discuss their semantics and equivalence, and demonstrate that they are suitable for a class of rasterization optimizations based on selective pre-rendering.

INTRODUCTION

In transportation systems, the information displays that may influence the vehicle's driving are considered safety-critical. Special efforts shall be made to ensure that these user interfaces are defect-free. Data-driven scene graphs have been introduced in (Esterel Technologies, 2007;[START_REF] Boisgérault | EDONA/HMI -modelling of advanced automotive interfaces[END_REF] as a class of models that are adapted to this task: they are composed of a functional description and a graphics description using the SCALABLE VEC-TOR GRAPHICS format (SVG) in our reference implementation. This structure is flexible enough to describe adequately safety-critical user interfaces but has enough constraints to enable validation to be performed at the modelling step. Most defects are therefore eliminated before the code generation stage that actually produces an executable software component.

Efficiency in the graphics rendering is a major issue for such mobile user interfaces that often rely on hardware with limited capabilities. We show in this article that the same type of structuring information that allows validation of data-driven scene graphs also enables optimization to be performed at the model level. Considerable efforts have already been done to design efficient SVG renderers. They either focus on specific hardware architectures [START_REF] Kim | Hardware implementation of a tessellation accelerator for the OpenVG standard[END_REF] or optimize some processing steps of software renderers [START_REF] He | Accelerated rendering of vector graphics on mobile devices[END_REF]. Our approach does not compete but rather complements these efforts as we want first and foremost to address the common situation where the complete graphics platform is already given. In this situation, the only choice left is to optimize the user interface model itself. We also notice that even if some optimizations may be applied to the graphics platform, once a data-driven scene graph model has been transformed into a display list, some model information has been lost. For example, graphics change detection is simple to perform on data-driven scene graphs models (see section 3) but difficult at a later processing stage [START_REF] Concolato | Design of an efficient scalable vector graphics player for constrained devices[END_REF]. We present a theoretical framework that enables automatic and safe optimization of data-driven scene graphs.

MODEL OF COMPUTATION

The data-driven scene graph Model of Computation consists of a hierarchical description of graphics components driven by a synchronous program. Synchronous programming languages are domainspecific languages dedicated to the design of real-time embedded systems. Their semantics are based on the synchronous hypothesis stating that the execution of a program is discretely divided into atomic reactions. In a reaction cycle, input signals are read/sampled, and internal computation takes place until outputs signals are emitted in answer, and the program state is progressed. In addition to the pure synchronous model, our model consumes its outputs to produce graphics.

The Synchronous Model

Register Transfer Level (RTL) description is a way of describing the operation of a synchronous digital circuit. Formally, a RTL description is a triplet (S 0 , ∆, Θ) that defines relations between the flows of inputs I k ∈ I , the flows of states S k ∈ S and the flows of outputs O k ∈ O of the RTL design. The transition function ∆ defines the next state of the circuit with respect to the input and the current state of the circuit:

S k+1 = ∆(I k , S k ) ∀k ∈ N
The output function Θ defines the output flow with respect to the input and the current state of the circuit:

O k = Θ(I k , S k ) ∀k ∈ N

The Graphics Model

As a graphics extension of the synchronous model, a data-driven scene graph model H ∈ H is formally defined as a quadruplet H = (S 0 , ∆, Θ, Γ) where (S 0 , ∆, Θ) is the pure synchronous part of the model and Γ ∈ G is the graphics function that computes scene graphs G k with respect to the output of the synchronous model:

G k = Γ(O k ) ∀k ∈ N
The scene graph is then rendered as an image using precise semantics rules. The EDONA/HMI model (Human Machine Interface -Work Package 4, 2008;[START_REF] Boisgérault | SVG for automotive user interfaces[END_REF] is our implementation of data-driven scene graphs. It is an extension of SVG, a family of specifications for describing twodimensional vector graphics. The data of the graphics model (geometric or style properties of graphics objects) may be exposed as variables while the document structure remains static.

Graphics Tree Grammar

The formal abstract syntax of the scene graph model that captures the SVG model is the following:

t ::= {n=v : t} | v ?t,t | t;t | g[n, ...n] | t e
with g ranging over graphics primitive names, n ranging over graphics properties names, v ranging over constant values or output signal names and e ranging on environments.

• {n=v : t} local assignment of graphics properties.

• v ?t 1 ,t 2 if v then t 1 else t 2 .

• t 1 ;t 2 compositing of t 2 over t 1 .

• g[n 1 , ...n k ] rasterization of the graphics primitive g using graphics properties n 1 . . . n k explicitely.

• t e is the raster image that results from the rendering of the graphics tree t in the environment e. This construct defines a unique raster image only when t is static -that is when the local assignments in t refer to constant values.

We write T the set of trees matching this grammar. We need to distinguish the special cases of group opacity and local transformation matrix. Therefore we will use the notations α v.t and µ v.t as syntactic sugar respectively for:

{opacity=v : t} and {matrix=v : t}

Display List

At each reaction, the data-driven scene graph is reduced into a display list matching the grammar:

t ::= g(c, ...c) | t;t | α c.t | µ c.t
with g ranging over graphics primitive names and c ranging over constant values and we write T * the set of display lists matching this grammar.

Semantics Rules

The reduction of a tree t ∈ T into a display list t ′ ∈ T * , given an environment E and the outputs O of the synchronous model at a given instant is written:

E, O ⊢ t ⇀ t ′
This reduction obeys the following semantics rules:

E[n ← O[v]], O ⊢ t ⇀ t ′ E, O ⊢ {n=v : t} ⇀ t ′ O[v] = 1 E, O ⊢ t 1 ⇀ t ′ 1 E, O ⊢ v ?t 1 ,t 2 ⇀ t ′ 1 O[v] = 0 E, O ⊢ t 2 ⇀ t ′ 2 E, O ⊢ v ?t 1 ,t 2 ⇀ t ′ 2 E, O ⊢ t 1 ⇀ t ′ 1 E, O ⊢ t 2 ⇀ t ′ 2 E, O ⊢ t 1 ;t 2 ⇀ t ′ 1 ;t ′ 2 ∀i ∈ [1..k] E[n i ] = v i E, O ⊢ g[n 1 , ...n k ] ⇀ g(v 1 , ...v k ) e, ∅ ⊢ t ⇀ t ′ E, O ⊢ t e ⇀ t ′
Graphics Equivalence Given a RTL model or a data-driven scene graph model:

H = (S 0 , ∆, Θ) or H = (S 0 , ∆, Θ, Γ)
if for all k ∈ N and for all input I k ∈ I we have:

O k = Θ(I k , S k ) and S k+1 = ∆(I k , S k )
and if for all environment E ∈ E we have:

E, O k ⊢ t 1 ⇀ t ′ 1 and E, O k ⊢ t 2 ⇀ t ′ 2 then: t ′ 1 = t ′ 2 ⇒ t 1 H ≡ t 2

SELECTIVE PRE-RENDERING

We propose to optimize the rendering time of data-driven scene graphs by using selective prerendering techniques. We want to replace branches of the graphics tree by bitmap images only when this replacement is indeed relevant. Considering our model, it is not always possible to apply this technique on any branch since some of the graphics parameters are re-computed at each reaction: as an example, consider a shape whose color depends on the value of a signal. On the other hand, if the position (x, y) of a shape is the sole changing property, then it is possible to save the shape in a bitmap and then combine the bitmap at the expected position.

When possible, it is not always advised to replace a branch by an equivalent bitmap. As an example consider the figure 1 depicting 2 simple shapes: on the left picture, shapes are quite big with respect to the bounding box and it might be useful to cache such a picture. On the right picture, the 2 shapes are much smaller and far distant: for the same bounding box, the image is quite empty. In this second case, the pre-rendering technique will probably be less efficient than the direct rendering of the image.

Minimisation Problem

In our approach, the performances of a model only depends on the graphics operations. The cost of the synchronous part of the system is supposed to be null.

We then define the cost function of the system on the graphics tree. Given a model H ∈ H together with its graphics tree t ∈ T and a cost function C : T → R + we want to compute a tree t 0 ∈ T equivalent to t that minimizes C , i.e. such that:

t 0 H ≡ t and C (t 0 ) ≤ C (t ′ ) ∀t ′ H ≡ t

The Cost Function

Computation time or space is consumed at each instant during the rasterization of the display list. Computation costs have 2 different origins:

1. the rasterization cost of graphics primitives 2. the compositing cost of bitmaps.

The global cost function C (t) is obtained by computing the mean of the instantaneous cost C i over each reaction of the system:

C (t) = ∑ k C i (E 0 , O k ,t)
The exact cost function is then highly dependent on the input of the considered data-driven scene graph, which prevent us from optimizing the design before it is run. Instead, we use an estimated cost function that is independent on the inputs of the system. This estimated cost can be a theoretical approximation of the graphics primitives costs or even computed on the fly using a sample of representative ouputs of the considered model.

Minimization Algorithm

The function that minimizes the cost of a tree t 0 using the selective pre-rendering technique is the function C (subst(t 0 ,t, t E )) < C (subst(t 0 ,t, M ′ (E,t))) then M (E,t) = t E else M (E,t) = M ′ (E,t) and the auxiliary function M ′ is defined as follows:

M : E × T → T. Let
M ′ (E, {n=v : t}) = {n=v : M (E[n ← ∅[v]],t)} M ′ (E, v ?t 1 ,t 2 ) = v ? M (E,t 1 ), M (E,t 2 ) M ′ (E,t 1 ;t 2 ) = M (E,t 1 ); M (E,t 2 ) M ′ (E, g[n 1 , ...n k ]) = g[n 1 , ...n k ] M ′ (E, t e ) = t e where ∅[v] = ⊥ if v is not a constant.
We tested the algorithm on a graphics interface representing the reconfigurable instrument cluster of a car: this includes lights, alert and warning indicators, odometer, speed, RPM, gas and temperature gauges. This model is a typical use case of safety-critical automotive user interface. This graphics interface is shown in figure 2. In this model, every indicator can be either "on" (coloured) or "off" (dark grey), furthermore, the red alert indicators can be scaled big and shown transparent over the speed/RPM gauges (as an emphasis for serious alerts). The odometer is made up of individual led bars (coloured of black) and any needle can rotate around its axis. The evolution of the pre-rendering algorithm is shown in the graphics of the figure 3. The x-axis represents the steps of the algorithm and the y-axis represents the number of frame per seconds (FPS) that the rasterization were able to achieve.

The cost function were here evaluated experimentally on a 32-bits linux based system equiped with a ATI Mobility Radeon graphics card and using the "image" software rendering backend of the cairo graphics library. Performances were evaluated on a representative set of input values that may influence the rendering performances of the model. The red line represents the evolution of the best performances of the model and the blue dots represent the performances of the current candidate (red line is the "max" of the blue dots).

CONCLUSION

We presented a theoretical framework for the modelling of graphics interfaces. We illustrated the usefulness of this framework with a method for an automated optimization of graphics performances. This method optimizes the rendering time of dynamic vector graphics by using a selective pre-rendering technique as evidences show that the systematic prerendering is generally not optimal. Groups of vector objects are thus replaced by equivalent bitmaps. This method relies on a cost function of primitive rasterization functions. This includes the cost of group opacity and matrix positioning operations. The global cost function can be evaluated on the fly during the minimization algorithm using a sequence of representative outputs. Experiments show the usefulness of our approach on a concrete safety-critical automotive instrument cluster model. The optimization algorithm benchmarks score a significant improvement of the rendering performances.

Figure 1 :

 1 Figure 1: Usefulness of Pre-rendering?

  cache?(E,t) be the function returning 1 if the tree branch t in the environment E can be cached, 0 otherwise. Let subst(t, b 1 , b 2 ) be the function that returns the tree t ∈ T where the branch b 1 ∈ T has been replaced by the branch b 2 ∈ T. Let t 0 be the root of the graphics tree and O 1 , ...O k be the output sequence of the model. The function M is defined as follows. If: cache?(t) ∧

Figure 2 :Figure 3 :

 23 Figure 2: The car's instrument cluster model.


Matthis Synofzik

Christian Beetz

Claudia Bauer

Michael Bonin

Elena Sanchez-Ferrero

Tanja Schmitz-Hübsch

Ullrich Wuellner

Thomas Nägele

Olaf Riess

Ludger Schöls

Keywords: diagnostic assessment, frequency, and phenotypic features

come

Reply to the Editor and the Reviewers

Dear Prof. Polychronakos, Thank you for the expedited review process and for giving us the opportunity to revise and resubmit our manuscript. The comments of the editors and the reviewers' comments were valuable for optimizing our manuscript. Please find attached a largely rephrased submission of the manuscript and a detailed response to the points raised by you and each of the two reviewers.

Sincerely, Ludger Schöls

Response to the Editor: " Specifically, the superiority of MLPA over qPCR should be better documented, per the comments of one of the reviewers."

Reply: After weighing all valuable comments of the reviewers we decided to remove our main qPCR data from the manuscript, following in particular the suggestion of reviewer #1. This decision was not easy as we have invested a lot of resources in the qPCR part. However, given the particularities of our DNA sample which includes partially old and degraded DNA from a continuous series of patients with dominant ataxia collected over almost 15 years, a direct comparison between qPCR and MLPA does indeed not seem fair and might lead to unjustified conclusions, as pointed out by the reviewers. Certainly, the challenges of difficult samples (partially old and degraded DNA) and large diagnostic demands (ideally measuring all exons at once) favor multiplexed diagnostic approaches like MLPA. We have learned that this fact is too trivial to exemplify with our questionable qPCR data. It has not been our intention to question qPCR for gene dosage analysis as a method itself, and we are convinced -like both reviewers suggest -that there is no intrinsic technical superiority of MLPA compared to qPCR. Thus, all remaining questions were carefully answered with a special focus on the validity of our MLPA assay, without comparison to qPCR.

Response to reviews: REVIEWER 1:

1. My main criticism relates to the justification for including the qPCR data. Clearly this particular qPCR assay is unsuitable and I don't see what useful information it adds. It may give the unfair impression that no qPCR assay would be suitable. Reply: We are grateful for this very convincing remark. For the reasons stated above, we omitted the qPCR data.

Page 5:

The MLPA probe sequences should be provided as supplementary data to allow others to replicate the findings." Reply: We now provide the MLPA probe sequences, reference MLPA probes for other genomic regions and size distribution of MLPA probes in the ITPR1 assay in supplementary table [START_REF] Schols | Autosomal dominant cerebellar ataxias: clinical features, genetics, and pathogenesis[END_REF]. Additionally, we provide an annotation of the MLPA probes in the revised figure 2.

Page 7:

There is no presentation of MLPA data. Given that this is an in-house developed assay, there needs to be some evidence that the MLPA assay is valid other than using the array method (which wasn't used on cases giving a normal MLPA result). Figure 1 is fine as a summary but I suggest presenting supplementary data or even presenting a single case in a figure including error bars indicating the normal and abnormal ranges and p-values for each deletion." Reply: We now provide MLPA data from control subjects including the normal variation, exemplary MLPA data of a single case compared to controls, means and standard errors of the means (in an extension of Figure 1) and quantitative MLPA data of all subjects in a table format (supplementary table 2) .. [START_REF] Edener | Early onset and slow progression of SCA28, a rare dominant ataxia in a large four-generation family with a novel AFG3L2 mutation[END_REF]. Some description of the SNP coverage of the ITPR1 exons would be useful other than the total number. Any deletion containing fewer than 10 SNPs would be difficult to detect. How many SNPs are contained in each of the deleted cases?" Reply: A new table (table 1) is introduced detailing deletion sizes and the number of SNPs contained in each SCA15 deletion.

5.

Where is F06 data in Figure 2?" Reply: F06 DNA was too old for accurate SNP array measurements (technical failure). A comment is given in table 2 description. [START_REF] Storey | A new dominantly inherited pure cerebellar ataxia, SCA 30[END_REF]. "More detail on breakpoints within SUMF1 is needed; Figs 1 and 2 are very uninformative on this. I take it that there was only array data on SUMF1; is the SNP coverage adequate to determine the SUMF1 breakpoints with confidence?" Reply: Details about the algorithms used to qualify CNVs have been included in the methods section. As far as possible the breakpoints in SUMF1 have been included in figure 2.

7. "'the third mutation (better say deletion)…..did not include the 3 prime of the ITPR1 gene' -please state which case this refers to. Furthermore, no deletion appears to include the 3 prime end." Reply The deletion in F48 does not include the 3 prime. We added the family codes to the deletions to allow a better identification and phrased the sentences more clearly. Reply: The acronym "ADCA" (=autosomal-dominant cerebellar ataxia) has been replaced by the acronym SCA (=autosomal-dominant spinocerebellar ataxia), which is explained in the introduction and used throughout the manuscript.

"

The discussion of balancing the resources against the accuracy of the test is good. Perhaps it is worth adding that for individual tests versus testing a cohort, a microarray approach would be best." Reply: We added this statement to the discussion.

12. "The statement that MLPA is 'superior' to qPCR shouldn't be made. Without wanting to offend, qPCR is not the easiest of techniques and in the hands of another laboratory might be satisfactory." Reply: The reviewer is certainly right. As already indicated in response to the first remark, the qPCR failures observed in our study were probably indeed attributable not to intrinsic deficits of qPCR, but to low DNA quality. Thus, a fair comparison between both methodologies does not seem possible, and we therefore omitted the qPCR data from the manuscript.

13. "Grammatical corrections: Page 5: 'qPCR rather 5' ITPR1…..' delete rather; Page 6: 'where' should be 'were'." Reply: We corrected both errors.

REVIEWER 2: 14. The superiority of MLPA vs qPCR from a technical point of view is not proved to me. In particular the authors need to improve the methods section, and verify that qPCR failures are not attributable to variants in case of a suspected deletion, technical problems or bad DNA quality." Reply: We completely agree that DNA quality is a critical factor for quantitative measurements, and the qPCR failures might indeed be attributable to low DNA quality, but not deficits in qPCR per se. Therefore, we omitted the qPCR data from the manuscript as stated above.

15. Numeric values are given for qPCR only in table 1, while they should be shown for both with standard deviations. Samples test both with qPCR and MLPA need to be repeated and DNA checked in case of strange results deletion/duplication in the same sample, values unexpected), but it is unclear if they were. Sample F29-051329 and F18-SCA1258 have strange values in qPCR that may be due to bad DNA quality. What about MLPA in these samples, values are missing? Sample F29-031401 results may be explained by two polimorphisms in heterozygosis in the tested exons, that need to be sequenced. Reply: We omitted the qPCR data from the manuscript. Numerical values are now given for MLPA, including standard deviations (figure 1; supplementary table 2). For sample F48-051329 (we think there was a type-o from the reviewer's side as we did not have a sample F29-051329; he probably intended to refer to F48-051329 as this sample had a falsely high value in qPCR), MLPA yielded decreased values, indicating a deletion (see current Figure 1 and supplementary table 2). For sample F18-SCA1258 (which was a false positive in qPCR), MLPA yielded normal results. Likewise, sample F29-031401 (which was also a false positive in qPCR) also yielded normal results in MLPA. In the present version of the manuscript these parts have been removed.

Figures do not emphasize results obtained and need a profound and careful revision."

Reply: We profoundly revised all our figures. 17. Table 1. qPCR data need to be expressed with a standard deviation, telling how many experiments for each exon were performed. The complete set of exons tested can be shown, at least in supplementary. The mean for different exons is a nonsense, because a deletion can be partial." 21. "Other points: Introduction, page 4, line 7. The authors cite SCA20 and SCA30 as "private" genes. Because these loci do not have a gene yet, and large screenings could not be done, they cannot be considered "private". Bibliography citations are lacking for loci/genes not reported in reference 1 [START_REF] Schols | Autosomal dominant cerebellar ataxias: clinical features, genetics, and pathogenesis[END_REF])".

Reply: See response to comment# 25 for qPCR methodology. The following table provides standard deviations of qPCR data (without means, as these are indeed not needed, as pointed out by the reviewer). This table was omitted in the current version of the manuscript:

Reply: We are grateful for this remark. SCA20 and SCA30 are now no longer referred to as "private genes" and we added bibliography citations that were lacking so far.

22. "Introduction, page 4, line 9. "less common SCA subtypes": again this is an assumption not supported by genetic screenings. Actually, the conclusions of the paper show that SCA15 is not so rare. I would suggest to change into "recently identified".

Reply: This is a good suggestion, which we adopted in the new version of the manuscript.

23. "Introduction, page 4, line 13 and throughout the texts. Numbers in the papers are generally in letters up to ten, and numerals from 11 on. Exception is at the beginning of a sentence (see page 5 line 8, "50%" should be "Fifty percent"." Reply: We revised the manuscript accordingly.

24. "Page 5, molecular genetics analyses, line 3. I do not understand the sentence: "For both MLPA and qPCR rather 5' ITPR1 targets were selected". Please reformulate." Reply: We rephrased this sentence as follows: MLPA targets were selected to cover especially 5' portions of the ITPR1 gene due to the fact that all deletions reported previously included at least exons 1 to 10.

25. "Page 5. Supplementary figure 1 is never cited in the text. It could be inserted in materials and methods. Page 6 line 3. Analysis of MLPA data, and below. It would be easier for the reader to find in supplementary materials all the information needed to set up the methods described, including primers, probes, PCR conditions, and methods used in the analysis of data (MLPA, and qPCR). It is not reported even the mix used for qPCR. Was a probe used or SYBR green for qPCR?" Reply: All details about the MLPA probes and qPCR primers are now given in supplementary table 1. Most parts of the qPCR results were now omitted from the manuscript. We only use qPCR to address the effect of the unusual preservation of the 5' end in one SCA15 family. More details on the qPCR method were added to the "material and methods" section. 40. An possible anticipation is visible in their families in supplementary figure1. it is curious anticipation in SCA15 was recently described by Di Gregorio et al., 2010. Is there a difference between different deletions, and in particular when SUMF1 is deleted?" Reply: This is a very interesting question. However, given the fact that SCA15 starts very slowly -and can even start "unnoticed" (as pointed out for two of our subjects)-the impression of "anticipation" might simply be due to the fact that current generations are more sensitive to even subtle coordination changes, which had not been paid attention to in their parental generations. We strongly believe that only larger patient cohorts and in particular prospective studies, starting early in the life course of asymptomatic SCA15 riskpersons, can truly investigate whether there is an anticipation effect in SCA [START_REF] Klebe | New mutations in protein kinase Cgamma associated with spinocerebellar ataxia type 14[END_REF]

Competing Interest:

None to declare.

Introduction

Spinocerebellar ataxias (SCAs) are a clinically and genetically heterogeneous group of autosomal dominant cerebellar ataxias with an overall prevalence of 3/100,000 [START_REF] Schols | Autosomal dominant cerebellar ataxias: clinical features, genetics, and pathogenesis[END_REF][START_REF] Van De Warrenburg | Spinocerebellar ataxias in the Netherlands: prevalence and age at onset variance analysis[END_REF]. Up to now, 28 SCA loci have been identified, whereby more than 60% of all SCAs are caused by CAG repeat expansions in the SCA1, SCA2, SCA3, SCA6 and SCA7 genes [START_REF] Schols | Autosomal dominant cerebellar ataxias: clinical features, genetics, and pathogenesis[END_REF]. Patients with autosomal-dominant ataxia who are negative for these mutations present a diagnostic challenge as the remaining SCA mutations include very low frequency genes (e.g. SCA11 [START_REF] Bauer | Spinocerebellar ataxia type 11 (SCA11) is an uncommon cause of dominant ataxia among French and German kindreds[END_REF] or SCA28 [START_REF] Edener | Early onset and slow progression of SCA28, a rare dominant ataxia in a large four-generation family with a novel AFG3L2 mutation[END_REF]) and gene candidates that still warrant further confirmation (e.g. SCA20 [START_REF] Knight | A duplication at chromosome 11q12.2-11q12.3 is associated with spinocerebellar ataxia type 20[END_REF] or SCA30 [START_REF] Storey | A new dominantly inherited pure cerebellar ataxia, SCA 30[END_REF]). To guide cost-and time-efficient genetic screening, more information about frequency, phenotypic characteristics and optimal diagnostic strategies in recently identified SCA subtypes is highly warranted.

SCA15 was recently found to be caused by multi-exon deletions and, more rarely and still debated, by missense mutations in the ITPR1-gene (inositol 1,4,5-triphosphate receptor, type 1) [START_REF] Van De Leemput | Deletion at ITPR1 underlies ataxia in mice and spinocerebellar ataxia 15 in humans[END_REF][START_REF] Hara | Total deletion and a missense mutation of ITPR1 in Japanese SCA15 families[END_REF][START_REF] Iwaki | Heterozygous deletion of ITPR1, but not SUMF1, in spinocerebellar ataxia type 16[END_REF][START_REF] Ganesamoorthy | Development of a Multiplex Ligation-Dependent Probe Amplification Assay for Diagnosis and Estimation of the Frequency of Spinocerebellar Ataxia Type 15[END_REF]. The phenotype of SCA15 has been studied in only relatively few patients as only 9 SCA15 families have been identified worldwide [START_REF] Ganesamoorthy | Development of a Multiplex Ligation-Dependent Probe Amplification Assay for Diagnosis and Estimation of the Frequency of Spinocerebellar Ataxia Type 15[END_REF][START_REF] Gregorio | Two Italian families with ITPR1 gene deletion presenting a broader phenotype of SCA15[END_REF], and the frequency of SCA15 in the European population is still unknown. In addition, the most efficient diagnostic method remains to be settled as several different molecular-genetic techniques have been used to detect ITPR1 deletions -in particular deletion-specific PCR [START_REF] Iwaki | Heterozygous deletion of ITPR1, but not SUMF1, in spinocerebellar ataxia type 16[END_REF], different DNA microarrays [START_REF] Van De Leemput | Deletion at ITPR1 underlies ataxia in mice and spinocerebellar ataxia 15 in humans[END_REF][START_REF] Hara | Total deletion and a missense mutation of ITPR1 in Japanese SCA15 families[END_REF] or multiplex ligation-dependent probe amplification (MLPA) [START_REF] Ganesamoorthy | Development of a Multiplex Ligation-Dependent Probe Amplification Assay for Diagnosis and Estimation of the Frequency of Spinocerebellar Ataxia Type 15[END_REF]. We here determined the frequency and phenotypic spectrum of SCA15 by screening a large cohort of SCA families negative for common SCA repeat expansions for deletions in the ITPR1-gene by a customized MLPA assay.

Material and Methods

Patient selection and assessment

A consecutive series of 274 German families with autosomal dominant transmission of ataxia was recruited from ataxia clinics in Bochum, Bonn and Tübingen (= all SCAs). Out of this sample, we selected those index patients for SCA15 screening who were negative for repeat expansions causing SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, SCA10, SCA12, SCA17, and DRPLA and for mutations causing SCA11, SCA14, and SCA27, yielding a cohort of 60 index patients (= unexplained SCAs). Four of these patients were excluded from the study due to low quantity and/or insufficient quality of the respective DNA samples. Mean age of onset of the remaining index patients (n=56) was 43.8 +/-17.4 years (range: 3-71). 50% (28/56) of the index patients presented with pure cerebellar ataxia and 50% with non-cerebellar signs like gaze palsy, epilepsy, spasticity, dystonia, Parkinsonism or peripheral neuropathy.

All index patients and, if available, further affected family members were assessed (i) by a clinical examination through a movement disorder specialist (M.S., L.S.) to identify cerebellar and non-cerebellar features, (ii) by the Scale for the Assessment and Rating of Ataxia (SARA) [START_REF] Schmitz-Hubsch | Scale for the assessment and rating of ataxia: development of a new clinical scale[END_REF] to determine severity and progression of ataxia, (iii) by magnetic resonance imaging (MRI) and (iv) by electrophysiological studies (including nerve conduction studies, motor evoked potentials, and sensory evoked potentials).

Molecular genetic analyses

In a first step, multiplex ligation-dependent probe amplification (MLPA) was used to screen for ITPR1 deletions. We designed a MLPA assay targeting the gene's promoter as well as exons 2, 8, 18, 41 and 54 and intron 4 with one probe each (=7 probes total). These targets were selected to cover especially 5' portions of the ITPR1 gene as all deletions reported previously included at least exons 1 to 10 [START_REF] Ganesamoorthy | Development of a Multiplex Ligation-Dependent Probe Amplification Assay for Diagnosis and Estimation of the Frequency of Spinocerebellar Ataxia Type 15[END_REF][START_REF] Gregorio | Two Italian families with ITPR1 gene deletion presenting a broader phenotype of SCA15[END_REF]. Six probes targeting different chromosomes were included for reference (ITPR1 MLPA probe sequences, reference MLPA probes for other genomic regions, and size distribution of MLPA probes in the ITPR1 assay are given in supplementary table 1). Pertinent oligonucleotides were obtained from MWG-Biotech (Germany) and reagents were taken from the EK1 kit offered by MRC-Holland (The Netherlands); they were applied according to the manufacturers' instructions. Analysis of MLPA data was carried out in analogy to previously described MLPA analyses [START_REF] Schule | Analysis of CYP7B1 in non-consanguineous cases of hereditary spastic paraplegia[END_REF].

In a second step, high-density SNP array was used for validation purposes and determination of the size of the deletion in all index cases for which MLPA suggested the presence of a heterozygous deletion. Samples were evaluated by an Affymetrix 6.0 SNP array platform using copy number variation (CNV) and analyzed with the Affymetrix CN4 algorithms within GTC v3. The predicted copy number as well as the start and end of the each CNV segment were determined using the Hidden Markov Model (HMM) incorporated in the software package using the Viterbi algorithm. For the ITPR1-SUMF1 locus approx. 380 markers were looked up for genomic imbalances as indicated by a reduction or amplification of hybridization signal (CNV) or excess of homozygosity. For the mathematical computation of the CNV sizes a sliding window method is used.

To investigate the potential effect of the 5' end on expression of the ITPR1 gene in family F34, a quantitative real time PCR (qPCR) was designed for amplicons in exon 3 (including the start codon) and exon 6 of the ITPR1 gene. Primers are given in supplementary table 1.

PCR amplification was performed in triplicates using a LightCycler480 system (Roche, Mannheim, Germany) with 10 μL PCR volumes in the 384-well block format using a standard qPCR protocol (15 min. 95°C, 45 cycles with 20 sec. 95°C, 40 sec. 55°C, and 20 sec. at 72°C, followed by a melting curve from 40°C to 85°C) with hotstart SYBRgreen chemistry (Roche, Mannheim, Germany). We established comparable amplification efficiencies for ITPR1amplicons and reference amplicons within the BCMA, SDC4, and B2MG genes using the relQuant analysis tool of the LightCycler software version 1.5. A reduction in qPCR signal by < 70% was defined as indicating a deletion.

Results

Genetic screening

MLPA suggested partial ITPR1 deletions in 5/56 index patients, but provided no evidence for multiplications (Figure 1; for detailed individual quantitative MLPA data, see supplementary table 2). Pedigrees of SCA15 families are provided in supplementary figure 1. In family F34 the deletion did not affect the 5' end of the ITPR1 gene: MLPA in all 3 affected family members revealed that this ITPR1-deletion preserved the promoter region and exon 2 of 1 and supplementary table 2).

ITPR1 (figure
In 4 of 5 index patients, heterozygous genomic ITPR1 deletions were confirmed by copy number analysis by SNP array. In these patients 176 to 383 markers were included in the deletion regions. Deletions -as measured by CNV-spanned approximately 183 kb to 423kb

(for details see table 1 and figure 2). The size of one deletion (F06-III-8) could not be determined by SNP-array analysis as compromised DNA quality did not allow accurate measurements (technical failure). Deletions in F39, F48 and F49 affected partly both the ITPR1-and the SUMF1-gene, without including the 3' prime of the ITPR1-gene (figure 2). In family F34, CNV array analysis did partially differ from MLPA results by predicting the border of the ITPR1-deletion about 10kb upstream of that defined by the MLPA markers (figure 2).

Since MLPA data in all affected members of family F34 consistently suggested that the deletion starts between exon 2 and intron 4 we designed a qPCR with primers covering the ATG triplet in exon 3 of ITPR1. In all three affected members of family F34 qPCR indicated the start codon to be deleted (qPCR fragment localization are shown in figure 2).

The ITPR1 deletions co-segregated with the disease in kindreds F34 and F48 (figure 1). In F06 and F49 no further family members were available for genetic testing. In F39, the father of the index patient had deceased and no DNA was available, but pertinent clinical records confirmed affection by cerebellar ataxia.

Phenotype and disease progression

Detailed clinical, electrophysiological and imaging characterisation was available for ten patients and is summarized in Tables 2 and3. Exemplary videos are available as supplementary material. In these ten patients, SCA15 presented clinically with a mid-life onset of 44.6 years (range 30-67 years) and a rather uniform phenotype of slowly progressive cerebellar ataxia (~1.2 SARA points/year, range 0.7-1.7; Tab. 2). A walker or wheelchair was needed around 15-17 years after disease onset. In 9/10 individuals the initial symptom was gait disturbance. Interestingly, 3 subjects (F34-IV-3, F34-IV-4, F48-III-1) considered themselves to be asymptomatic despite unequivocal signs of cerebellar ataxia and cerebellar atrophy on MRI (figure 3, c-f). Two of them had been clinically diagnosed for more than 5 years. This fact again highlights the subtle and slow progression of the disease.

As a characteristic feature, action and postural tremor of the hands and partly of the head were found in 7/10 patients. It aggravated the intention tremor which was consistently observed in all subjects. Subjects perceived tremor as one of the most compromising impairments for activities of daily living. In 3/7 subjects action and postural tremor evolved rather late in the disease course (14-17 years after ataxia onset; Table 2). Other extracerebellar features included clinical or electrophysiological signs of pyramidal tract affection (4/10 patients), electrophysiological evidence of dorsal column pathology (4/10 patients) and upward gaze palsy (1/10 patients) (Tables 2 and3). Two patients showed psychiatric symptoms already before the onset of ataxia (paranoid-hallucinatory psychosis in F34-IV-3; adjustment disorder in F39-III-4), one elderly patient revealed mild cognitive impairment (F49-III-3) presumably due to vascular encephalopathy. None of the patients showed facial myokymias or buccolingual dyskinesias which have recently been reported in some SCA15 patients [START_REF] Gregorio | Two Italian families with ITPR1 gene deletion presenting a broader phenotype of SCA15[END_REF]. In line with previous findings [START_REF] Gardner | Spinocerebellar ataxia type 15[END_REF], MRI demonstrated a rather uniform pattern of cerebellar atrophy affecting mainly the vermis (in particular superior and mid-vermal regions), with only minor hemispheric involvement and sparing of brainstem and cerebral structures (figure 3).

Atrophy seemed to progress only slowly within 4 and 5 years of follow-up, respectively (figure 3, g-j, m-p). However, no data for quantitative volumetric analysis were available.

Discussion

In our series SCA15 is relatively frequent, accounting for 8.9% (5/56) of unexplained SCAs and 1.8% (5/274) of all SCAs. This finding exceeds a recent, also MLPA-based estimate of 2.7% ITPR1 deletions in Australian SCA cases negative for repeat expansions [START_REF] Ganesamoorthy | Development of a Multiplex Ligation-Dependent Probe Amplification Assay for Diagnosis and Estimation of the Frequency of Spinocerebellar Ataxia Type 15[END_REF]. It clearly exceeds the reported prevalence of other "new" SCA subtypes like SCA14 [START_REF] Klebe | New mutations in protein kinase Cgamma associated with spinocerebellar ataxia type 14[END_REF], SCA11 [START_REF] Bauer | Spinocerebellar ataxia type 11 (SCA11) is an uncommon cause of dominant ataxia among French and German kindreds[END_REF],

SCA27 [START_REF] Brusse | Spinocerebellar ataxia associated with a mutation in the fibroblast growth factor 14 gene (SCA27): A new phenotype[END_REF] or SCA28 [START_REF] Edener | Early onset and slow progression of SCA28, a rare dominant ataxia in a large four-generation family with a novel AFG3L2 mutation[END_REF]. Thus, SCA15 seems to present the fifth most common SCA subtype after SCA1, 2, 3 and 6 and is even more frequent than SCA7, 8 and 17 in the German population.

Our customized MLPA assay allowed for the consistent measurement of seven target regions distributed over the ITPR1-gene, demonstrating that each family in our series had a unique deletion. Moreover, it revealed that SCA15 can also be caused by heterozygous deletions that spare the first 2 non-coding exons, while previous studies always demonstrated inclusion of ITPR1-exon 1 in SCA15 deletions [START_REF] Hara | Total deletion and a missense mutation of ITPR1 in Japanese SCA15 families[END_REF][START_REF] Ganesamoorthy | Development of a Multiplex Ligation-Dependent Probe Amplification Assay for Diagnosis and Estimation of the Frequency of Spinocerebellar Ataxia Type 15[END_REF]. Whereas MLPA (and qPCR) are locus specific tools for measuring gene dosage, SNP array copy number analysis has to rely on serial data points in order to size variations. This might explain why the deletion genotyped in all three SCA15 patients of family F34 resulted in consistent results for MLPA (and qPCR), but not for CNV arrays: although the telomeric border was identical for all patients, it did not combine with our MLPA data, and the centromeric border varied for almost 15.000 bases. Moreover, for the mathematical computation of deletion sizes in the CNV array a sliding window method was used, which frequently leads to over-estimation of the deletion size (in our case the questionable region is covered by only 9 SNPs). Correspondingly, since MLPA data consistently demonstrated the promoter region and exon2 of ITPR1 to be preserved in all affected members, we believe that the CNV array method indicated too large genomic deletion regions. To address the effect of the unusual preservation of the 5' end we designed a qPCR system, which demonstrated inclusion of the ITPR1 start codon in exon 3 by the deletion. Thus, no proper protein translation can take place and this deletion resembles all other deletions by entailing IPTR1 haploinsufficiency.

Variability of deletion size as found in our and other studies would argue for high resolution deletion detection systems that completely interrogate all ITPR1-exons. This fact also indicates that our screening by MLPA might even underestimate the true SCA15 prevalence, as this approach may miss small deletions of exons not covered by the MLPA probe-sets used in this study. Moreover, this approach cannot reliably detect point mutations in the ITPR1 gene. So far, a single amino acid change of unknown significance is reported in one patient (c.8581C>T, p.P1059L; [START_REF] Hara | Total deletion and a missense mutation of ITPR1 in Japanese SCA15 families[END_REF]). The frequency of ITPR1 point mutations and small deletions in SCA families, however, seems to be very low, as they have not been found in 38 SCA families that were negative for large ITPR1 deletions and common SCA mutations [START_REF] Van De Leemput | Sequencing analysis of the ITPR1 gene in a pure autosomal dominant spinocerebellar ataxia series[END_REF].

Although our study recommends MLPA as a cost and time-efficient screening tool for SCA15, for individual tests rather than testing of a cohort, a DNA microarray approach might still be most efficient.

In a comprehensive phenotypic characterization of our series of SCA15 patients we confirmed findings of earlier reports with smaller patient numbers [START_REF] Gregorio | Two Italian families with ITPR1 gene deletion presenting a broader phenotype of SCA15[END_REF][START_REF] Gardner | Spinocerebellar ataxia type 15[END_REF][START_REF] Hara | Japanese SCA families with an unusual phenotype linked to a locus overlapping with SCA15 locus[END_REF] This finding, however, needs to be confirmed in prospective studies with iterative SARA evaluations as retrospective estimation of disease onset in such a slowly progressive disease course is difficult. Disabling action and postural tremor, found in 70% of our patients, seems to be characteristic of (albeit not specific to) SCA15 [START_REF] Hara | Japanese SCA families with an unusual phenotype linked to a locus overlapping with SCA15 locus[END_REF]. It may present as the initial symptom (see patient F48-III-7 and [START_REF] Gardner | Spinocerebellar ataxia type 15[END_REF]), or develop more than a decade after onset of ataxia.

Intriguingly, all patients (100%) demonstrated cerebellar atrophy affecting mainly the superior and mid-vermal regions, with only minor hemispheric involvement and sparing of the brainstem and cerebral structures. This atrophy pattern may occur even before subjectively noticed ataxia (patients IV-3 and IV-4 of family F34).

Contrary to earlier perspectives which considered SCA15 to be a pure cerebellar ataxia [21],

extra-cerebellar features such as pyramidal tract affection, dorsal column pathology or gaze paresis might be associated with SCA15 as well (as they are in many other SCAs [START_REF] Schols | Autosomal dominant cerebellar ataxias: clinical features, genetics, and pathogenesis[END_REF]), yet present in a more variable fashion.

In conclusion, we show that SCA15 presents the most common non-trinucleotide repeat SCA in Central Europe. MLPA seems to serve as a robust and cost-effective screening tool for

ITPR1

Licence for publication statement:

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  8. "Fig 2 should be redrawn to focus more on the ITPR1 and SUMF1 regions. Most of the figure as it stands is wasted space. Reply: We have completely changed figure 2, now focussing more on ITPR1 and SUMF 1 regions. 9. "Page 8, last sentence, first para: this is a repeat of what has been stated earlier in the paragraph." Reply: To avoid this redundancy, we omitted the statement earlier in the paragraph. 10. "Page 10: the acronym ADCA needs explained."

1 )

 1 43. "Legends to the figures. Fig.1. "seven localizations" change for instance to "seven regions" . Check: "Red bars visualize the least length". At the end indicate the name of the five families among brackets." Reply: We corrected the errors and indicated the names of the five families.44. Fig.2. Title:Change to " Copy number variation…" .First sentence change to: "Copy number ….SCA15 region for three sample of family F34 (…), F39-051019, F48-0551329, and F49-050994 are shown." Erase next line. "F06-SCA0142 …" and last line. These sentences are not useful here, leave this comment in materials and methods. Additionally …." change to "The sample F29-031401 was suggestive for a heterozygous ITPR1 gene deletion based on qPCR results, but normal in MLPA." Reply: The figure legend has been modified accordingly. Videos are part of ms Original Article Spinocerebellar ataxia type 15: diagnostic assessment, frequency, and phenotypic features Matthis Synofzik, M.D 1,6* ; Christian Beetz, PhD 2* , Claudia Bauer 3 ; Michael Bonin, PhD 3 , Elena Sanchez-Ferrero 2 , Tanja Schmitz-Hübsch, M.D. 4 ; Ullrich Wüllner, M.D. 4 ; Thomas Nägele, MD 5 , Olaf Riess, M.D. 3 ; Ludger Schöls, M.D. 1,6#, Peter Bauer, M.D. 3 Department of Neurology, Hertie-Institute for Clinical Brain Research, Neurodegenerative Diseases (DZNE), University of Tübingen, Germany *Both authors contributed equally to this study. # Corresponding author: Dr. Ludger Schöls, Clinical Neurogenetics, Department of Neurology and Hertie-Institute for Clinical Brain Research, University of Tübingen, Hoppe-Seyler-Str.

Figure 1 :

 1 Figure 1: MLPA readouts for all SCA15 patients with heterozygous ITPR1 gene deletions. (A) MLPA analysis allowed for the relative gene dosage measurements in seven localizations throughout the ITPR1-gene: hybridization probes have been placed in the promoter, in exon 2, intron 4, exon 8, exon 18, exon 41 and at the 3 prime end in exon 54. Red bars visualize the minimal length of heterozygous ITPR1-deletions and demonstrate at least four different deletion sizes in our five families. (B) Exemplary MLPA data. Mean and SEM of healthy controls (n=15) and SCA15 patients (3 (exon 54) to 9 (introns 4 and 8) observations; see also part (A)). Moreover, the relative peak area for patient F34-III-2 is shown in grey bars for the respective fragments. Values around 1.0 indicate presence of the normal two copies whereas values around 0.5 suggest a heterozygous deletion. (chr: chromosome; prom: promoter; ex: exon; in: intron; for calculation of relative signal value see Material and Methods section).

Figure 2 :

 2 Figure 2: Copy number variation analysis for six SCA15 patients with heterozygous ITPR1 gene deletions. Copy number plots of the relative gene dosage in the SCA15 region for three samples of family F34 (F34-III-2, F34-IV-3, F34-IV-4), F39-III-4, F48-III-7, and F49-III-3 are shown. F06-III-8 gave low quality results due to degraded DNA (not shown). A ruler with genomic coordinates and the relative position of the ITPR1 and SUMF1 gene is shown. All individual deletion sizes (as determind by SNP array CNV) are shown as horizontal bars and labelled with the family code. Moreover, the exact position of all MLPA probes (promoter and exon 2 are very close to each other) and of the qPCR fragments is shown.

Figure 3 :

 3 Figure 3: Brain imaging of SCA 15 patients. MRI and CT brain scans of 7 SCA15 patients demonstrating a highly uniform picture of prominent cerebellar atrophy affecting mainly the vermis (in particular the superior and mid-vermis), with only minor cerebellar hemispheric involvement and complete sparing of the brainstem and cerebral structures. MRI brain scans of patients F39 III-4 (g-j) and F48 III-7 (m-p) were assessed two times within 5 years and 4 years, respectively, indicating minor progression of vermal cerebellar atrophy. Left columns: sagittal view; right columns transversal view; a,e: T2-weighted images; b, h, f, j, l, n, p, r: T2 FLAIR; g, i, k, m, o, q: T1 flash sequences; c,d: CT scans. Patient IV-3 of

Table 1

 1 The scheme of the genes, below needs to be prepared in a more detailed way and not only copied and past from Ensembl. Indicate better the positions and the hgs used; where are centromere and telomere located? What is the scale on the left? What are the shadows (specify in the legend)." Reply: Figure2has been modified according to the suggestions of the reviewer. The extension of the deletions and involved exons as determined by SNP array are now given in

	:
		ID	Exon 3	Exon 6
		F06-SCA0142 =	Failed	0.542±0,146
		F06-III-8
		F34-040232 =	0.559±0,116	0.379±0,069
		F34-III-2
	SCA15	F49-III-3 F49-050994 =	0.388±0,108	0.583±0,297
		F39-051019 =	0.403±0,115 (a)	0.645±0,328
		F39-III-4
		F48-051329 =	0.645±0,293	1.403±0,293 (b)
		F48-III-7
	false-positives	F18-SCA1258 F29-031401	0.152±1,023 (a) 0.450±0,089 (a)	1.600±0,391 0.575±0,109 (a)
	qPCR data points represent mean relative gene dosage changes normalized to reference gene
	dosages (see method section). In three samples a total of four false positive measurements
	was observed (indicated by (a) ): F39-051019 (exon 1 qPCR values were reduced, whereas
	MLPA demonstrated normal values for promoter and exon 2 probes); F18-SCA1258 (one
	extremely reduced data point in exon 1; the data point in exon 4 was normal) and F29-031401
	(two apparently consistent measurements in exon 1 and 4). Neither MLPA analysis nor high
	density SNP array analysis (not performed for sample F18-SCA1258) yielded suspicious
	signals in these three samples. Moreover, qPCR analysis (and high density SNP array
	analysis) failed for exon1 in sample F06-SCA0142 due to bad DNA quality (this sample had
	been stored for almost 12 years) and sample F48-051329 was normal for exon 4 (false -
	negative; indicated by (b) ).
	18. "Note to table 1 contain comments that should not be here. Notes in tables do not contain
	comments on the results. Put only the relevant information to read values, leave comments in
	the results section."
	Reply: The former table 1 is now completely removed.
	19. "Figure 1. The real MLPA data, or at least the calculations need to be showed. A more
	elegant scheme of the gene and the tested exon should be provided. See for instance the figure
	reported in Ganesamoorthy et al., 2009 (reference 6). In this paper it is clear that also MLPA
	has a great variability. This figure may contain a second panel with the data of table 1. The
	location of the regions tested by qPCR and MLPA could be given in a unique scheme."
	Reply: We now provide detailed MLPA data in figure 1 and, in particular, in supplementary
	table 2. Calculations are performed as already reported earlier (Schüle et al., 2009;
	Neurogenetics), this reference is referred to in the manuscript. A clarified scheme of the
	genomic region with coordinates of all deletions, MLPA and qPCR locations is now given in
	figure 2.
	20. "Figure 2. It is important to clearly show the extension of the deletions found. I propose to
	zoom the figure to a region more close to ITPR1 gene. Add for instance a double arrow to
	each SNP array plot with the extension of the deletion. Specify, the likely minimal-maximal
	extension of the deletion in Kb for each deletion found, defining the breakpoints from the
	SNP-array probes. It is never cited which are the involved exons in ITPR1 and SUMF1 (when

table 1 .

 1

.

  28. "Page6, line 15. "For nine index patients…": this part is not materials and methods and should be put in results. Because the authors emphasize the superiority of MLPA on qPCR I think that more details are needed. Which gene was used as reference (BCMA, SDC4, B2MG, all three?)? What the authors did when a sample had a suspected deletion/duplication? Did they repeat the test, and how many times? It is important to run a group of controls to verify the variability of the gene dosage in normal subjects; was this done? If not, how the authors define the threshold of 70% for deletions? Was DNA quality / degradation verified when a sample was found as a false positive? For qPCR and MLPA standard deviations need to be shown." Reply: For qPCR methodology, please refer to the response to comment #25. Standard errors of the means (SEM) for MLPA are now given in figure1.29. "Page 6 and throughout the text. Check all gene names are in italics (see for example lines 11 and 14)." Reply: The manuscript has been revised accordingly.37. "qPCR results showing a possible deletion may be explained by a variant in that exon. The exon needs to be sequenced." Reply: The respective qPCR data are now omitted.38. "Page 8, line 3. The authors are assuming MLPA provided accurate results. This is a circular reasoning. In the discussion they conclude MLPA give more reliable results, but it seems because the authors already though it was more reliable." Reply: MLPA showed different deletions for different families, but yielded highly reliable results within each family, thus indicating its reliability. All ITPR1 deletions detected by MLPA were confirmed by an independent SNP array approach and also MLPA estimates of minimal deletion sizes were confirmed by SNP array. This indicates the validity of our MLPA assay. However, we admit that we cannot exclude false negative findings with our MLPA assay, as stated clearly in the discussion. Such findings, however, would even increase the relatively high frequency of SCA15 in patients with unexplained SCA, which we report in our study.

	35. "Page 7 line 19. "The smallest deletion…" change to "The smallest deletion was found in
	patient F49, and partly …". Next line "which was slightly larger in the ITPR1 gene". The
	sentences need to be reformulated, with a more precise value. I would advise the authors not
	to use generic terms."

26. "Page 6. Cite the reference accession number of the ITPR1 gene used. Numbering of exons can change with time. Reply: The reference sequence (NM_001099952) is now given in supplementary table

1.

27. "Page 6, line 21. "More than 120 SNPs…": the authors are too generic. They do know how many, and their location." Reply: More precise numbers of SNPs are now provided in the method section and table 130. Page 6 line 15. "qPCR signals were augmented": it is necessary to be more specific; a value and a standard deviation need to be provided. How many times the experiments were done?" Reply: See comment 17 and comment 25.

31. "Page 6 line 17, and throughout the text. "exemplarily" should be changed. The entire paragraph can be simplified. The authors tested seven patients with a possible deletion and two out of nine patients with a possible duplication. I would not add further explanations that may results in a circular reasoning." Reply: "Exemplarily" was omitted with the main qPCR data from the manuscript.

32. Page 6 line 20. "represented by eight data points". The points tested in ITPR1 are reported to be seven in page 5, and in legend to figure

1

. Reply: The correct number is indeed seven. We corrected the error on page 6.

33. "ID numbers are not homogeneous throughout the text and in figures. Sometimes a subject is cited with its Family-DNA code, sometimes with the number in the pedigree." Reply: This is a valid comment. We now used the subject codes from the pedigrees (supplementary figure) throughout the manuscript and omitted DNA-codes.

34. "Page 7 line 18. "The deletion spanned approximately": is it possible to define the exons involved?" Reply: Details of deletions are now presented in table 1. Its relation to the SUMF1 gene is shown in the revised figure

2

as far as the accuracy of the SNP array approach allows. Reply: We are very grateful for this remark. We modified the paragraph according to this suggestion and tried to omit generic terms as far as possible.

36. "Page 7 last sentence: "This finding, which could be confirmed…". I think is "This finding, which was confirmed…". Reply: We corrected the sentence accordingly. 39. Page 9. "The five families …. for genetic testing". This part can be simplified. The discussion can be shortened." Reply: The discussion has been shortened accordingly.

  deletions. Screening for ITPR1 deletions should be particularly performed in patients with unexplained SCA presenting with slowly progressive ataxia, prominent tremor and pronounced vermal atrophy of the cerebellum. Storey E, Gardner RJ, Knight MA, et al. A new autosomal dominant pure cerebellar ataxia. Neurology 2001;57(10):1913-5.

Table 1 : SNP array analysis in SCA15 patients

 1 Detailed deletion sizes and affected SNPs are given according to the Hidden Markov Model (HMM). Patient F06-III-8 was not included because compromised DNA quality did not allow accurate SNP-array analyses (technical failure).

	ID	Size Markers Start	End	Start / Marker	End Marker
					5' UTR ITPR1	3' UTR ITPR1
	F34 III-2 423kb	383 4521773 4944763	SNP_A-8328224	SNP_A-8319160
					5' UTR ITPR1	3' UTR ITPR1
	F34 IV-4 407kb	377 4521773 4928818	SNP_A-8328224	SNP_A-2196964
					5' UTR ITPR1	3' UTR ITPR1
	F34 IV-3 408kb	379 4521773 4929877	SNP_A-8328224	CN_1044140
					Intron 3 SUMF1	Intron 6 ITPR1
	F39 III-4 206kb	196 4477165 4683436	SNP_A-1940542	SNP_A-8427137
					Intron 2 SUMF1	Intron 20 ITPR1
	F48 III-7 219kb	212 4493662 4712980	SNP_A-2122078	CN_1033009
					Intron 3 SUMF1	Intron 4 ITPR1
	F49 III-3 183kb	176 4484080 4667436	SNP_A-1898571	SNP_A-1805189

Table 2 : Clinical findings in SCA15 patients.

 2 Scale for the Assessment and Rating of Ataxia, maximum score 40, minimum score 0. Disease progression is calculated by SARA score last examination / disease duration. ** SARA item gait, maximum score 8, minimum score 0. # Disease duration was not determinable in F48 III-1 as she was subjectively still unaffected and no previous assessment had been performed before. Ages and durations of symptoms in years.

	Fami	pedigree	Age at	age of onset	disease	initial	gait ataxia	limb	Dysarthria	dyspha	eye movements		pyramidal	postural/	intention	other features
	ly	number	last			progressio	features	(SARA item	dysmetria		gia			features	action	tremor
			exami			n		gait) **							tremor
			nation	Gait	Postural	(SARA						Nystagmus	saccade
				distur	tremor	Score							s
				bance		/disease
						duration) *
		III-2	80	60	74	24/20=1.2	gait	7	+(UE and	++	+	Horizontal +	Hyperm	n	++	+++ (UE &	n
							disturbanc	(wheelchair	LE)			vertical	etric		(UE&LE)	LE)
							e	with 75 yrs				gaze-evoked
								of age)
		IV-3	46	41	n.a.	6/5=1.2	dizziness	2	± (only	n	n	transient	hypo-	n	+ (UE)	+ (UE	hyporeflexia
									UE)			horizonal	and			&LE)	LE; transient
												gaze-evoked	hyperm				paranoid-
	F34												etric				hallucinatory psychosis (22yrs of age)
		IV-4	51	46	n.a.	5.5/5= 1.1	gait	2	±(only	n	n	n	hypo-	n	n	+ (LE)	n
							disturbanc		LE)				and
							e						hyperm
							(subjectiv						etric
							ely still
							not
							noticed)
		II-3	62	45	50	"slow"	gait	n.a.	+	+	+	horizonal	Hypom	+ (	+ (UE)	+ (LE)	n
							disturbanc	(walking	(UE+LE)			gaze-evoked	etric	extensor
							e	frame with						plantar
								62 yrs of						response,
								age)						but not
	F39	III-4	50	35	49	16.5/15=1	gait	3	+	n	n	n	n	spasticity) n	+ (head,	+	upward gaze
						.1	disturbanc		(UE+LE)						UE, LE)		palsy;
							e										adjustment
																	disorder
		II-3	69	50	67	21.5/19=	gait	4 (partial use	+	++	+	Horizontal +	hypo-	n	+ (UE;	+(UE)	n
						1.1	disturbanc	of crutches	(UE+LE)			vertical	and		trunk)
	F48						e	with 64 yrs of age)				gaze-evoked	hyperm etric

n n=none; ± questionable; n.a.= not available; + =mildly abnormal/affected; ++= moderately abnormal/affected; +++= severely abnormal/affected; UE= upper extremity; LE=lower extremity. *SARA,

Table 3 : MRI and electrophysiology data

 3

	fam	pedigree	MRI	MEP	SEP	NCS	Other
	ily	number
		III-2	cerebellar atrophy	n	not evoked	n	n
			(vermis> hemispheres)
	F34	IV-3	(vermis> hemispheres) (CCT) cerebellar atrophy	n	not evoked	n	n.
		IV-4	cerebellar atrophy	n	n	n	n
			(vermis> hemispheres)
		II-3	cerebellar atrophy	n.a.	n.a.	n.a.	n
			(vermis> hemispheres)
	F39	III-4	cerebellar atrophy (vermis> hemispheres)	legs: not evoked;	n	n	normal electronysta
				arms:			gmography
				normal
		II-3	cerebellar atrophy	legs: not	prolonged	n	normal
			(vermis> hemispheres)	evoked;	latency		cognitive
				arms:	bilateral		state
				normal			(MMSE
							29/30)
	F48	III-1	(vermis> hemispheres) cerebellar atrophy	n.a.	n.a.	n.a.	n
		III-7	cerebellar atrophy	n	n	n	n
			(vermis> hemispheres)
		III-3	cerebellar atrophy	n	prolonged	tibial	normal VEP
			(vermis> hemispheres)		latency	nerve:	and BAEP;
	F49				bilateral	reduced amplitude; sural	mild cognitive impairment
						nerve:	(MMSE
						normal	24/30)
		III-8	superior cerebellar	n	n	n	normal
			vermis atrophy				sympathetic
	F06						skin response,
							VEP and
							BAEP

Acknowledgements

We are grateful to the family members for their participation. This work was funded by the European Union (to the EUROSCA consortium; LSHM-CT-2004-503304) and the Stiftung für Pathobiochemie und Molekulare Diagnostik (to C.B.). MS was supported by a grant from the Volkswagen Stiftung (Az. II/85158).

Abstract

Background: To guide time-and cost-efficient analyses of the increasing number of autosomal-dominant spinocerebellar ataxia genes (SCAs), more information about frequency distributions, phenotypic characteristics and optimal diagnostic strategies is warranted.

Objective: To assess the prevalence and phenotypic spectrum of SCA15 and to confirm multiplex ligation-dependent probe amplification (MLPA) as a robust and efficient strategy for routine molecular diagnosis.

Methods: Fifty-six German SCA families negative for common repeat expansions were