BACKGROUND

A novel oncogenetic clinic was established in 2002 at the Royal Marsden NHS Foundation Trust offering advice and specialist follow-up for families with a germline mutation in BRCA1 or BRCA2. The remit of this multidisciplinary clinic, staffed by individuals in both oncology and genetics, is to provide individualised screening recommendations, support in decision-making, risk-reducing strategies, cascade testing, and an extensive research portfolio.

METHODS

A retrospective analysis was performed to evaluate uptake of genetic testing, riskreducing surgery and cancer prevalence in 346 BRCA1/BRCA2 families seen between January 1996 and December 2006.

RESULTS

661 individuals attended the clinic and 406 mutation carriers were identified; 85.8% mutation carriers have chosen to attend for annual follow-up. 70% of mutation carriers elected for risk-reducing bilateral salpingo-oophorectomy (RRBSO). 32% of unaffected women chose risk-reducing bilateral mastectomy; 32% of women with breast cancer chose contralateral risk-reducing mastectomy at time of diagnosis. Some women took over eight years to decide to have surgery. 91% of individuals approached agreed to participate in research programmes.

INTERPRETATION

A novel specialist clinic for BRCA1/2 mutation carriers has been successfully established. The number of mutation positive families is increasing. This and high demand for RRBSO in women over 40 is inevitably going to place an increasing demand on existing health resources. Our clinic model has subsequently been adopted in other centres and this will greatly facilitate translational studies and provide a healthcare structure for management and follow up of such people who are at a high cancer risk.

INTRODUCTION

Clinical testing for BRCA1 and BRCA2 germline mutations through the National Health Service (NHS) became available in the United Kingdom (UK) in 1995-6. Whilst the population frequency of BRCA1 and BRCA2 mutations is not high, collectively they account for approximately five percent of breast and ovarian cancer, moreover the risks of these cancers are high in mutation carriers; with lifetime risks of an estimated 44-75% for breast cancer and 20-45% for ovarian cancer in BRCA1 and 10-20% in BRCA2 mutation carriers. [START_REF] Antoniou | Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case Series unselected for family history: a combined analysis of 22 studies[END_REF][START_REF] King | Breast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2[END_REF] An increased cancer risk is also recognised in men with BRCA2 mutations, conferring a six percent lifetime risk of breast cancer and a three to seven fold increased risk of developing prostate cancer. [START_REF] Cancer | Cancer risks in BRCA2 mutation carriers. The Breast Cancer Linkage Consortium[END_REF][START_REF] Liede | Cancer risks for male carriers of germline mutations in BRCA1 or BRCA2: a review of the literature[END_REF] Increasing demand for BRCA1/2 gene mutation testing from individuals with a family history of breast and/or ovarian cancer, coupled with advances in molecular diagnostics, has led to a rapid rise in the number of BRCA gene mutation carriers identified. However, there are no national or international guidelines for the long-term clinical management of carriers. For mutation carriers, there are a number of recommendations focussing on medical management, including surveillance programmes and risk-reducing surgery, but there are currently no national guidelines regarding the best means of maintaining contact with families. Ongoing contact is important for providing up-to-date information about changes in management, supporting decision-making, and disseminating information through the family.

Research is changing the way in which these patients are managed with the development of new modalities for early detection, an increased understanding of the impact of environmental and reproductive factors on modification of risk and with advances in chemoprevention and treatment options. [START_REF] Antoniou | Reproductive and Hormonal Factors, and Ovarian Cancer Risk for BRCA1 and BRCA2 Mutation Carriers: Results from the International BRCA1/2 Carrier Cohort Study[END_REF][START_REF] Rebbeck | Effect of short-term hormone replacement therapy on breast cancer risk reduction after bilateral prophylactic oophorectomy in BRCA1 and BRCA2 mutation carriers: the PROSE Study Group[END_REF][START_REF] Andrieu | Pregnancies, breast-feeding, and breast cancer risk in the International BRCA1/2 Carrier Cohort Study (IBCCS)[END_REF][START_REF] Brohet | Oral contraceptives and breast cancer risk in the international BRCA1/2 carrier cohort study: a report from EMBRACE, GENEPSO, GEO-HEBON, and the IBCCS Collaborating Group[END_REF] The best means of updating families and their health professionals on screening and prevention recommendations in a field that is constantly changing remains uncertain. [START_REF] Mckinnon | Results of an Intervention for Individuals and Families with BRCA Mutations: A Model for Providing Medical Updates and Psychosocial Support Following Genetic Testing[END_REF] Different models of ongoing follow-up have been evaluated reporting mixed results. [START_REF] Karp | The prophylactic mastectomy dilemma: A support group for women at high genetic risk for breast cancer[END_REF][START_REF] Prospero | Psychosocial issues following a positive result of genetic testing for BRCA1 and BRCA2 mutations: findings from a focus group and a needs-assessment survey[END_REF][START_REF] Speice | Family issues in a psycho-education group for women with a BRCA mutation[END_REF] These studies indicate that there is a need for ongoing follow-up and contact with families with BRCA mutations, but have focused largely on supportive aspects of care rather than information provision and guiding decision-making. This is where a specialist multi-disciplinary follow-up clinic may be effective.

The aim of this study is to present our experience of a novel model for the follow-up of BRCA1/2 mutation carriers and perform a retrospective analysis of clinic attendance, uptake of risk-reducing surgery, research participation, pattern of genetic testing, and cancer occurrence in families who have attended the clinic over a ten year period.

METHODS

At the Royal Marsden NHS Foundation Trust (RMH), families harbouring a mutation in BRCA1 or BRCA2 have been offered annual clinical follow-up since the introduction of clinical testing in 1995. In 2002, as the number of families increased, follow-up was formalised into a novel oncogenetic clinic specifically for BRCA1 and BRCA2 families, called the Carrier Clinic.

The remit of this multidisciplinary clinic is to provide individualised screening recommendations and clinical management, ongoing psychological support, advice about cascade testing, and facilitate family communication. In addition it offers an opportunity for those at risk to engage in ongoing research programmes. The clinic is staffed by individuals with training in oncology and genetics, including medical practitioners, genetic/nurse counsellors and research staff. Clinic appointments are offered annually and for those individuals who find it difficult to attend the clinic a telephone consultation with a Clinical Nurse Specialist or Genetic Counsellor is offered. [START_REF] Eeles | Developments in Clinical Practice: Follow up Clinic for BRCA Mutation Carriers: a Case Study Highlighting the "Virtual Clinic[END_REF] This virtual clinic enables patients to have a point of contact in between appointments for ongoing help and support and provides a point of rapid access.

Bilateral risk-reducing mastectomy (RRM) or contralateral RRM with simultaneous reconstruction is an option discussed with all women with a known BRCA mutation without evidence of metastatic disease. A psychologist is involved in the assessment of unaffected women considering bilateral RRM. Risk-reducing salpingooophorectomy (RRBSO) is recommended on completion of child-bearing, usually from age 40 although women wishing to pursue this at an earlier age are referred to a gynaecologist for assessment. The use of hormone replacement therapy (HRT) in unaffected women who have ovarian surgery prior to the natural menopause is recommended up to 50 years to help maintain bone health and to avoid vasomotor symptoms in this group.

All families identified between July 1996 and December 2006 with a pathogenic germline mutation in BRCA1 or BRCA2 seen at RMH have been included in this study. A total of 346 families were seen (201 with BRCA1 mutations and 145 with BRCA2 mutations). Mutations in these families were identified either through the UK National Health Service (NHS) (n = 275), through the commercial vendor Myriad (www.myriad.com, n = 25) or through research testing which was subsequently confirmed in the diagnostic setting (n = 46). All mutation carriers and untested family members were offered annual follow-up in the clinic with. At each follow-up visit, data on mutation status, cancer occurrence, surveillance and risk-reducing surgery were collected.

Statistical Analysis

Descriptive statistics were obtained using SPSS statistical software (version 15.0).

RESULTS

A total of 661 individuals from 346 families were eligible for inclusion in this analysis (406 mutation carriers: 239 BRCA1, 165 BRCA2, two both BRCA1 and BRCA2; 193 individuals with a negative predictive test, and 62 untested family members) constituting the entire patient group that came forward for a consultation between 1996 and 2006.

Of the 406 mutation carriers 33 were male (16 BRCA1, 17 BRCA2) and 373 female (223 BRCA1, 148 BRCA2, 2 BRCA1 & BRCA2). At time of mutation detection 4 men and 172 women had not had a cancer diagnosis. The mean length of follow-up from the date of mutation detection was 48 months (range 0.5-127 months).

Genetic Testing

Between July 1996 and December 2006 406 individuals were identified with a pathogenic mutation in BRCA1 and/or BRCA2. Out of 188 mutations identified through direct mutation analysis, 35 were identified in the research setting. Four hundred and thirty individuals eligible for predictive testing have attended the clinic and 368 of these [308 women (84%) and 60 men (16%)] proceeded to testing. Median age at testing was 44 years (range 21-79 years). Figure 1 summarises the method of mutation detection in the 406 carriers and how this has changed over time. Time to predictive testing was calculated using the date the mutation was first identified in the family and the test result date [data on individuals referred from other centres for follow-up have been excluded (n=43)]. Median time to predictive testing was five months (mean 14.5 months; SD = 20.9; Range 1-121 months). Fifty of 368 individuals (13.6%) waited three years or more before proceeding to predictive genetic testing.

Year of Test

Of the 373 female BRCA1/2 mutation carriers, 85.8% chose to have regular follow-up either through the carrier clinic or by telephone. Table 1 summarises the follow up in tested and untested individuals. Table 2 describes the uptake into research programmes. Of the 661 members of families with a known BRCA gene mutation, invited to take part in research programmes the uptake was high with 91% of mutation carriers, 72% non-carriers and 74% untested individuals enrolled. 8) appointments in the carrier clinic were attended prior to surgery. Twenty-one women had a simultaneous hysterectomy.

Three incidental cancers were detected at RRBSO (see Table 4). In all cases no abnormalities had been detected on transvaginal ultrasound pre-operatively and CA125 levels had been within normal range.

DISCUSSION

The population frequency of BRCA1 and BRCA2 mutations in the UK are estimated at 0.12% and 0.20% respectively, with higher frequencies in certain ethnic groups such as the Ashkenazim. [START_REF] Antoniou | The BOADICEA model of genetic susceptibility to breast and ovarian cancers: updates and extensions[END_REF] We found our clinic population to consist of approximately two-thirds BRCA1 and one third BRCA2 mutation carriers which is likely to be explained by the chronology of the introduction of BRCA1 and BRCA2 mutation testing into clinical practice.

The number of gene carriers identified has rapidly increased in recent years in our clinic. This increase may in part be explained by the introduction of full sequencing of coding regions plus Multiplex Ligation-dependent Probe Amplification (MLPA) from early 2006 onwards and the availability of research testing, particularly for families that do not meet the criteria for NHS testing. This together with an increasing awareness of genetic testing for cancer predisposition is having an impact on demand for genetics services across the world. A large proportion of gene carriers was identified through predictive testing. We found the median time interval to predictive testing from the time the family were informed was five months, which is a shorter time to predictive testing than previously reported by other UK groups. [START_REF] Brooks | BRCA1/2 predictive testing: a study of uptake in two centres[END_REF][START_REF] Holloway | Uptake of testing for BRCA1/2 mutations in South East Scotland[END_REF]. However, a significant minority of subjects were found to defer testing by several years, which is in line with these reports, and may reflect more distant relatives waiting for a result in a closer relative before deciding to undergo testing or the decision to wait until an age is reached where screening or risk-reducing surgery become influencing factors [START_REF] Brooks | BRCA1/2 predictive testing: a study of uptake in two centres[END_REF][START_REF] Holloway | Uptake of testing for BRCA1/2 mutations in South East Scotland[END_REF][START_REF] Evans | Comparison of proactive and usual approaches to offering predictive testing for BRCA1/2 mutations in unaffected relatives[END_REF].

The results of this study show that there is a high demand for follow up within a formal clinic setting with over 85% of individuals choosing to either attend the clinic or have an annual telephone appointment. A high level of uptake of research programmes was observed, with over 90% of those people invited subsequently enrolled in a study. This may be facilitated by the face-to-face contact of the clinic as poorer response rates have been reported when comparing postal invitations with face-to-face recruitment.

Bilateral RRM reduces the risk of breast cancer by an estimated 90% and provides a significant reduction in mortality. [START_REF] Rebbeck | Bilateral prophylactic mastectomy reduces breast cancer risk in BRCA1 and BRCA2 mutation carriers: the PROSE Study Group[END_REF][START_REF] Hartmann | Efficacy of bilateral prophylactic mastectomy in women with a family history of breast cancer[END_REF][START_REF] Evans | Uptake of risk-reducing surgery in unaffected women at high risk of breast and ovarian cancer is risk, age and timedependent[END_REF] Twenty eight percent of unaffected women in our cohort elected to have bilateral RRM and, to date, no breast cancers have been reported post-surgery. Uptake levels in other single centre studies vary from 15-51%. [START_REF] Evans | Uptake of risk-reducing surgery in unaffected women at high risk of breast and ovarian cancer is risk, age and timedependent[END_REF][START_REF] Meijers-Heijboer | Presymptomatic DNA testing and prophylactic surgery in families with a BRCA1 or BRCA2 mutation[END_REF][START_REF] Scheuer | Outcome of preventive surgery and screening for breast and ovarian cancer in BRCA mutation carriers[END_REF][START_REF] Meijers-Heijboer | Use of genetic testing and prophylactic mastectomy and oophorectomy in women with breast or ovarian cancer from families with a BRCA1 or BRCA2 mutation[END_REF] A large international study combined data from 9 centres and found an overall uptake of RRM of 18% in unaffected women, but this varied between 36.3% in the United States (US) and 2.7% in Poland. [START_REF] Metcalfe | Hereditary Breast Cancer Clinical Study Group. International variation in rates of uptake of preventive options in BRCA1 and BRCA2 mutation carriers[END_REF] Therefore the variation reported is likely to reflect local practice and cultural differences. The median interval of nine months from genetic testing to bilateral RRM is longer than that reported in two studies, one from Europe and one from the US, and may reflect differing local surgical protocols or health system processes. [START_REF] Meijers-Heijboer | Presymptomatic DNA testing and prophylactic surgery in families with a BRCA1 or BRCA2 mutation[END_REF][START_REF] Scheuer | Outcome of preventive surgery and screening for breast and ovarian cancer in BRCA mutation carriers[END_REF] A recent evaluation of a large UK series reported similar findings to this analysis, with the majority of women choosing bilateral RRM within 2 years of receiving their genetic test result. [START_REF] Rebbeck | Bilateral prophylactic mastectomy reduces breast cancer risk in BRCA1 and BRCA2 mutation carriers: the PROSE Study Group[END_REF] We found that women attended an average of three appointments in the Carrier Clinic before proceeding to surgery, suggesting the importance of the multidisciplinary team in supporting decision making. Studies have demonstrated the absence of long-term psychological impact on body image or sexuality providing the decision has been made in the context of careful counselling. [START_REF] Frost | Long-term satisfaction and psychological and social function following bilateral prophylactic mastectomy[END_REF][START_REF] Hopwood | Clinical follow-up after bilateral risk reducing ('prophylactic') mastectomy: mental health and body image outcomes[END_REF] Contralateral breast cancer risk in carriers has been estimated at 50-60% by age 70. [START_REF] Cancer | Cancer risks in BRCA2 mutation carriers. The Breast Cancer Linkage Consortium[END_REF][START_REF] Easton | Breast and ovarian cancer incidence in BRCA1 mutation carriers. Breast Cancer Linkage Consortium[END_REF] Contralateral RRM has been found to reduce the risk of a second primary by 90-95%, however further data are required to establish whether it reduces breast cancer-specific mortality. [START_REF] Mcdonnell | Efficacy of contralateral prophylactic mastectomy in women with a personal and family history of breast cancer[END_REF][START_REF] Van Sprundel | Risk reduction of contralateral breast cancer and survival after contralateral prophylactic mastectomy in BRCA1 or BRCA2 mutation carriers[END_REF][START_REF] Evans | Screening for familial ovarian cancer: poor survival of BRCA1/2 related cancers[END_REF] One third of confirmed BRCA mutation carriers attending the Carrier Clinic chose to have contralateral RRM as a preventative measure as a part of their breast cancer treatment which is a similar proportion to that reported in a large international study (27•3%). [START_REF] Metcalfe | Hereditary Breast Cancer Clinical Study Group. Predictors of contralateral prophylactic mastectomy in women with a BRCA1 or BRCA2 mutation: the Hereditary Breast Cancer Clinical Study Group[END_REF] In our cohort approximately one fifth of women affected by breast cancer have had more than one primary breast cancer, with a small subset having multiple primary cancers. There is therefore a strong case for considering contralateral RRM at initial diagnosis for those in good prognostic groups from their primary disease, in this population. In particular, this may be an important consideration for women diagnosed under the age of 50 years as the residual breast cancer risk is thought to plateau over the age of 50. [START_REF] Antoniou | Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case Series unselected for family history: a combined analysis of 22 studies[END_REF][START_REF] Kauff | Risk-reducing salpingooophorectomy in women with a BRCA1 or BRCA2 mutation[END_REF][START_REF] Rebbeck | Prevention and Observation of Surgical End Points Study Group. Prophylactic oophorectomy in carriers of BRCA1 or BRCA2 mutations[END_REF] Women who have completed their family are informed about the significant benefits of having RRBSO prior to the natural menopause both to reduce ovarian cancer risk by 90% and breast cancer risk by approximately 50%. [START_REF] Kauff | Risk-reducing salpingooophorectomy in women with a BRCA1 or BRCA2 mutation[END_REF][START_REF] Rebbeck | Prevention and Observation of Surgical End Points Study Group. Prophylactic oophorectomy in carriers of BRCA1 or BRCA2 mutations[END_REF] [START_REF] Domchek | Mortality after bilateral salpingooophorectomy in BRCA1 and BRCA2 mutation carriers: a prospective cohort study[END_REF][START_REF] Ford | Genetic heterogeneity and penetrance analysis of the BRCA and BRCA genes in breast cancer families[END_REF] which could explain the difference in these results. Neither study present data using a cut-off of 40 years for comparison. The high proportion of women over 40 choosing RRBSO observed in this cohort could be due to the recent validation of data about the risk-reduction offered by this approach and the lack of data supporting the efficacy of ovarian screening as an alternative. [START_REF] Evans | Screening for familial ovarian cancer: poor survival of BRCA1/2 related cancers[END_REF][START_REF] Stirling | Familial Ovarian Cancer Screening: current protocols are ineffective in detecting early stage ovarian malignancy[END_REF] Three occult cancers were detected at surgery, of which two were early stage. All three women had normal TVUS and CA125 results immediately prior to surgery, and were enrolled in ovarian screening programmes. These results support the increasing evidence that screening is not a safe alternative to surgery. [START_REF] Evans | Screening for familial ovarian cancer: poor survival of BRCA1/2 related cancers[END_REF][START_REF] Stirling | Familial Ovarian Cancer Screening: current protocols are ineffective in detecting early stage ovarian malignancy[END_REF][START_REF] Gaarenstroom | Efficacy of screening women at high risk of hereditary ovarian cancer: results of an 11-year cohort study[END_REF].

The majority (61%) of women had RRBSO within a year of finding out their test results suggesting that this could be a strong motivation for testing. On average, two or three appointments in the carrier clinic were required to facilitate this decision, highlighting the role of this clinic in this decision-making process. Psychosocial research has shown that RRBSO is an emotionally significant procedure and that balanced information should be provided about the risk reducing benefits of surgery versus the potential physical and emotional consequences. [START_REF] Hallowell | High-risk premenopausal women's experiences of undergoing prophylactic oophorectomy: a descriptive study[END_REF] It is universally accepted that the fallopian tubes should be removed during surgery but there is on going debate about whether the uterus should also be removed, with practice varying between countries. The data about whether there is an increased risk of endometrial cancer in BRCA carriers are preliminary. [START_REF] Beiner | Hereditary Ovarian Cancer Clinical Study Group. The risk of endometrial cancer in women with BRCA1 and BRCA2 mutations. A prospective study[END_REF] If hysterectomy is performed then HRT post-RRBSO can be easier to manage using oestrogen only preparations. An argument for retaining the uterus is that it reduces the risk of complications and thus morbidity. In the UK usual practice is to remove the ovaries and fallopian tubes but not the uterus unless indicated by the individual's medical history. This practice would be supported by the opinions in the literature. [START_REF] Beiner | Hereditary Ovarian Cancer Clinical Study Group. The risk of endometrial cancer in women with BRCA1 and BRCA2 mutations. A prospective study[END_REF][START_REF] Lu | Does a BRCA mutation plus tamoxifen equal hysterectomy?[END_REF] The experience reported by some gene carriers is that the significance of their genetic status can get lost across specialities. The average General Practice will manage only a handful of gene carriers but for the majority of families, once seen by a genetics service, screening is organised locally. BRCA1 and BRCA2 account for an estimated five to ten percent of breast and ovarian cancer cases, constituting a small percentage of an oncologist's caseload (and arguably less than the estimated 5% as more women opt for preventative surgery). Genetic status is an important consideration when a cancer is diagnosed, for example the significant risk of a contralateral breast primary. In addition there is some suggestion that platinum agents are more effective in women with BRCA1 and BRCA2 mutations who are being treated for ovarian cancer than in sporadic ovarian cancer cases. [START_REF] Cass | Improved survival in women with BRCAassociated ovarian carcinoma[END_REF][START_REF] Tan | BRCAness" syndrome in ovarian cancer: a case-control study describing the clinical features and outcome of patients with epithelial ovarian cancer associated with BRCA1 and BRCA2 mutations[END_REF] With the advent of new targeted drug therapies such as PARP inhibitors currently being trialled in the research arena, knowledge of genetic status is going to become an increasingly important consideration. [START_REF] Fong | Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers[END_REF] It was found that structured follow up within the Carrier Clinic led to the earlier diagnosis of eight cancers. A high number of breast cancers were observed during follow-up, with 31 detected in 933 women years. In the NHS Breast Screening Programme the expected cancer detection rate over the same time period is 2.5 cancers. [START_REF]Breast Screening Programme[END_REF] In conclusion, this novel clinic enables a team of professionals with a specialist interest in cancer genetics to coordinate the management of these families. It facilitates rapid communication of changes in practice recommendations to patients.

A very high number of individuals choose follow up in this clinic and the number of families is increasing annually both through clinical and research testing. There is a higher demand for RRBSO reported amongst this cohort than previously reported and the clinic plays a significant role in facilitating decision-making. This clinic model has subsequently been adopted in two other centres in London, offering the opportunity to greatly facilitate translational studies in this group. There are currently no national or international recommendations about the best practice for following up of BRCA1/2 gene carriers; such guidance could help to direct funding sources to support these specialist clinics.

Figure 1 :

 1 Figure 1: Pattern of genetic testing from 1996-2006

Table 1 . Choice of follow-up

 1

	Choice of Follow Up	Gene	Untested
		carriers	(n = 62)
		(n = 373)
	Attends carrier clinic	296 (79•4%)	47 (75•8%)
	Annual telephone follow-up	24 (6•4%)	2 (3•2%)
	Local follow-up (moved out of area)	36 (9•7%)	1 (1•6%)
	Elderly / unwell	10 (2•7%)	0
	No follow-up	7 (1•9%)	12 (19•4%)

Table 2 . Participation in research Cancer Prevalence

 2 Of 406 male and female BRCA1/2 mutation carriers, a total of 322 cancers were diagnosed in 245 individuals. The cancer spectrum is described in Table3. In total 254 breast cancers were observed in 202 individuals. Thirty one of 115 (20.5%) BRCA1 and 14/88 (15.9%) BRCA2 carriers have had more than one primary breast cancer with median time to a second primary of five and six years respectively.

		Number of individuals	BRCA1	Mean age at first diagnosis	BRCA2	Mean age at first diagnosis
	No cancer	161	95	na	66	na
	Breast Cancer	202*	115	39•4 yrs (25-67)	88 +	43•6 yrs (21-71)
	Ovarian / Fallopian Tube Cancer	50	36	49•8 yrs (32-64)	14	56•2 yrs (46-74)
	Primary Peritoneal Cancer	3	2	46•0 yrs	1	52•0 yrs
	Prostate Cancer	4	1	48•0 yrs	3	50•7 yrs
	Other cancer	11	4	na	7	na

Thirty-three individuals (all female) have died during follow-up: 14

(

11 BRCA1; 3 BRCA2) died from metastatic breast cancer; 15 (11 BRCA1, 4 BRCA2) from metastatic ovarian cancer and four from other cancers. * Includes 1 woman with BRCA1 and BRCA2 mutations. + One individual was male. na: not applicable

Table 3 : Observed cancers in clinic cohort (includes all individuals diagnosed with cancer pre and post testing).

 3 were detected through investigation of symptoms elicited in clinic and one breast cancer was detected by initiation of an overdue screening test. This equates to 31 breast cancers being detected in 933 women years. Fifty-three of the 373 female BRCA1/2 mutation carriers had already undergone bilateral mastectomy at time of mutation detection (24 for bilateral breast cancer, 24 had contralateral RRM and five had bilateral RRM based on family history criteria). Of the 320 women with remaining breast tissue post mutation detection, bilateral RRM was chosen by 89 (27.8%) and 9 of 28 (32.1%) women who subsequently developed breast cancer chose contralateral RRM at time of treatment. Post mutation detection, unaffected women chose to have bilateral RRM at a mean age of 37.4 years (range 23-62yrs). The median interval from mutation detection to surgery was nine months (range 2-64 months). A mean of 2.8 (range 1-8) appointments in the Carrier Clinic were attended prior to surgery. Four out of 98 women were found to have an incidental cancer at RRM (4.1%). All had previously had breast cancer. The histopathology of these cancers is detailed in Table4.

	Forty-nine cancers developed in 45 individuals post mutation detection: 15 were
	detected through routine screening (13 breast cancers, one ovarian cancer, one
	prostate cancer) 19 were self-detected / symptomatic (nine breast cancers, six
	ovarian cancers, one prostate cancer, three other cancers) and seven were detected
	at time of risk-reducing surgery (four breast cancers and three ovarian cancers). Of
	the self-detected / symptomatic breast cancers, six were interval cancers and three
	were outside of screening. Of the self-detected / symptomatic ovarian cancers four
	were interval cancers and two were outside of screening.

An additional eight cancers were detected as a direct result of attendance at the Carrier Clinic. Four cancers (two breast cancers and two prostate cancers) were detected through research screening tests, three (two breast cancers, one primary peritoneal cancer)

Table 4 : Incidental cancers detected at risk-reducing surgery post-mutation detection

 4

	(insignificant details amended to maintain anonymity)
	Out of 373 female BRCA1/2 carriers 296 had ovarian tissue at the time of mutation
	detection. Seventy-seven had previously had their ovaries removed: 40 as treatment
	for ovarian cancer, three as treatment for breast cancer, three for benign
	gynaecological conditions and 31 had chosen RRBSO prior to knowledge of genetic
	status.
	Post mutation detection, RRBSO was chosen by 131/186 (70.4%) women aged over
	40 and 142/239 (59.

4%) women aged 35 and over (women with advanced metastatic breast cancer were excluded unless they had survived beyond two years). There was no significant difference in the mean ages of surgery for BRCA1 (46 years, SD = 7.5) and BRCA2 (48 years, SD = 7.7) carriers. Post mutation detection, the median time to surgery was 9 months (mean 16.7, range 0-104 months). A mean of 2.4 (range 1-

  RRBSO has been reported to reduce mortality in this high-risk population.[START_REF] Domchek | Mortality after bilateral salpingooophorectomy in BRCA1 and BRCA2 mutation carriers: a prospective cohort study[END_REF] A high proportion of women attending the Carrier Clinic chose RRBSO; 49% of women over 35 years and 70% of women over 40 years. Meijers-Heijboer et al.(2003) report similar results in a Dutch cohort with 49% of mutation carriers over the age of 35 choosing surgery.[START_REF] Meijers-Heijboer | Use of genetic testing and prophylactic mastectomy and oophorectomy in women with breast or ovarian cancer from families with a BRCA1 or BRCA2 mutation[END_REF] Kauff et al.(2002) reported that 58% of women over the age of 35 proceeded to surgery in one centre in the US.[START_REF] Kauff | Risk-reducing salpingooophorectomy in women with a BRCA1 or BRCA2 mutation[END_REF] However, in the US RRBSO is recommended from age 35, whereas in the UK this is usually offered from age 40 based on the BCLC penetrance estimates,

Acknowledgements

We thank Professor Nazneen Rahman at the Institute of Cancer Research, London and Professor Shirley Hodgson and her team at St George's Healthcare NHS Trust for referring patients to the Carrier Clinic.

Contributors

EK Bancroft had overall responsibility for the analyses and writing of the article. All authors contributed to the study design, provided data or contributed to data interpretation, writing, or editing of the report, and approved the final version. R. Eeles and A. Ardern-Jones developed the Carrier Clinic Model and R. Eeles had overall responsibility for the study.

Conflict of interest

RE has received an educational grant from Vista Diagnostics and support from Astra Zeneca for a feasibility cancer prevention trial.

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


Kathrin Schuldt

Claudia Esser

Jennifer Evans

Jurgen May

Christian Timmann

Christa Ehmen

Wibke Loag

Daniel Ansong

Andreas Ziegler

Jürgen May

Tsiri Agbenyega

Christian G Meyer

Rolf D Horstmann
email: horstmann@bnitm.de

FCGR2A Functional Genetic Variant Associated with Susceptibility to Severe Malarial Anemia in Ghanaian Children

Keywords: Falciparum malaria, malarial anemia, FcγRIIa, CD32, genetic variation

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Severe malaria caused by infection with Plasmodium falciparum remains one of the major health problems in sub-Saharan Africa.[1] It predominantly affects infants and young children and causes approximately 1 million fatalities each year.[2] Severe falciparum malaria is a complex clinical syndrome comprising a number of life-threatening conditions including severe malarial anemia, cerebral malaria, metabolic acidosis, respiratory distress and other, less frequent complications. [START_REF] Marsh | Indicators of life-threatening malaria in African children[END_REF] Severe anemia is the most frequent malaria complication among infants and young children living in endemic areas with very high transmission levels.

Its pathogenesis is considered complex and appears to include intravascular hemolysis, extravascular clearance of parasitized and non-parasitized erythrocytes, and bone-marrow dysfunction. In severe anemia the apparently rapid loss of a large proportion of erythrocytes greatly exceeds the proportion of erythrocytes that are infected by parasites. [START_REF] Woodruff | Cause of anaemia in malaria[END_REF][START_REF] Price | Factors contributing to anemia after uncomplicated falciparum malaria[END_REF] The mechanisms causing a loss of non-parasitized erythrocytes are still unclear but appear to involve immunological processes. [START_REF] Kai | The pathophysiology of malarial anaemia: where have all the red cells gone?[END_REF][START_REF] Jakeman | Anaemia of acute malaria infections in non-immune patients primarily results from destruction of uninfected erythrocytes[END_REF] IgG receptors (FcγR) are crucial mediators of humoral immune responses. FcγRIIa, a low-affinity IgG receptor, links humoral to cellular arms of the immune response by mediating phagocytosis of IgG-and CRP-opsonized structures. The receptor is expressed on various human cell types including macrophages, monocytes and neutrophils. It has been described to bind three of the four IgG subclasses and appears to be the only receptor for the IgG 2 isotype. Its binding capacity is critically influenced by a genetic variant (rs1801274; FCGR2A c.497A>G) where the substitution of an adenine (A) by a guanine (G) results in a replacement of a histidine (H) by an arginine (R) residue at amino acid position 131.

While FcγRIIa 131H binds IgG 2 efficiently, the structural variant FcγRIIa 131R was reported to hardly interact with IgG 2 and to have a decreased affinity for IgG 3 . [START_REF] Warmerdam | A single amino acid in the second Ig-like domain of the human Fc gamma receptor II is critical for human IgG2 binding[END_REF] Instead, FcγRIIa 131R was found to be the major receptor for CRP on monocytes and neutrophils. [START_REF] Stein | C-reactive protein binding to FcgammaRIIa on human monocytes and neutrophils is allele-specific[END_REF] Thus, FcγRIIa 131R not only results in a loss of function for IgG 2 and IgG 3 binding, but also includes a gain of function regarding the ability to bind CRP, an opsonizing serum protein of the innate immune response. [START_REF] Kb | FcgammaRIIa expression with FcgammaRI results in C-reactive protein-and IgG-mediated phagocytosis[END_REF] In contrast to the antigen-specific recognition by antibodies, binding of CRP is based on pattern recognition of altered self and foreign molecules. [START_REF] Clos | Function of C-reactive protein[END_REF] In infants, very high levels of CRP production have been measured during clinical malaria episodes. [START_REF] Hurt | Do high levels of C-reactive protein in Tanzanian children indicate malaria morbidity[END_REF] Opsonization by CRP causes phagocytosis and an inflammatory response by an induction of cytokine synthesis. These activities are mediated by the ability of CRP to bind to FcγR and to activate complement. FCGR genetic variants have been reported to influence human susceptibility to a variety of infectious and autoimmune diseases. [START_REF] Rascu | Clinical relevance of Fc gamma receptor polymorphisms[END_REF] The effect of FcγRIIa H131R on malaria has been addressed in several studies but the data are difficult to interpret. The H allele was found associated with protection against severe malaria in Sudanese adults [START_REF] Nasr | Fc gamma receptor IIa (CD32) polymorphism and antibody responses to asexual blood-stage antigens of Plasmodium falciparum malaria in Sudanese patients[END_REF] and FcγRIIa 131HH with protection against both severe and mild malaria in Indian adults. In contrast, the same variant was associated with susceptibility to severe malaria in Gambian children [START_REF] Sinha | Polymorphisms of TNF-enhancer and gene for FcgammaRIIa correlate with the severity of falciparum malaria in the ethnically diverse Indian population[END_REF][START_REF] Cooke | Association of Fcgamma receptor IIa (CD32) polymorphism with severe malaria in West Africa[END_REF], and FcγRIIa 131RR was reported to protect against high parasite densities in Kenyan children. [START_REF] Shi | Association of FCgamma receptor IIA (CD32) polymorphism with malarial anemia and high-density parasitemia in infants and young children[END_REF]18] With the objective to investigate the association of the FcγRIIa H131R variants with the various forms of severe falciparum malaria, we studied more than 1500 children with severe malarial anemia and more than 500 cases of cerebral malaria.

METHODS

Patients and Controls

The study was carried out in an area hyperendemic for falciparum malaria of the Ashanti Region of Ghana, West Africa. Patients were recruited at Komfo Anokye Teaching Hospital, a tertiary medical referral center in Kumasi, between 2001 and 2005 in parallel with the "Severe Malaria in African Children" study. All pediatric patients 6 months to 10 years of age were screened for malaria parasitemia using Giemsa-stained blood films. In malaria patients, the level of consciousness according to the Blantyre Coma Score (BCS) [1], blood glucose, lactate (YSI Life Sciences), and hemoglobin concentrations (Sysmex KX-21N) were determined at admission. Patients positive for asexual P. falciparum parasitemia with either a BCS of <3, hemoglobin concentration of <5 g/dL or lactate of >5 mmol/L [1] were enrolled after consent had been obtained from accompanying parents or guardians. Parasite densities were determined per 200 leukocytes and calculated assuming a leukocyte count of 8,000 per µl blood. [START_REF] Taylor | Standardized data collection for multi-center clinical studies of severe malaria in African children: establishing the SMAC network[END_REF] In the analysis patients with a hemoglobin level of < 5g/dl, a BCS of < 3, or any other than the two complications were classified as severely malarial anemic (n=1591), cerebral (n=562), and other (n=497), respectively. Within a total of 2504 cases, 146 patients were affected by both conditions, severe anemia and cerebral malaria. A detailed description of the participants, ethnicities and clinical diagnoses has been reported elsewhere. [START_REF] May | Hemoglobin variants and disease manifestations in severe falciparum malaria[END_REF] To serve as a control group, apparently healthy children were recruited through community surveys.

The study was approved by the Committee for Research, Publications and Ethics of the School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. All procedures were explained to parents or guardians of the participating children in the local language, and written or thumb-printed consent was obtained.

Genotyping

In order to extract DNA for subsequent genotyping, blood samples were drawn from all participants. 0.5-1 ml blood was collected into citrate, and, before subjected to DNA extraction, the granulocyte fraction obtained from density gradient centrifugation was preserved in 4-M urea. DNA was extracted according to the suppliers' instructions (Nucleo-Mag 96 Blood; Macherey-Nagel, Düren, Germany). Prior to genotyping whole-genome amplification (WGA) using 10 ng DNA of each sample was conducted according to manufacturer's instructions (GenomiPhi HY DNA Amplification Kit, GE Healthcare, Braunschweig, Germany).

Statistical analyses

Genotype frequencies were tested for deviation from Hardy-Weinberg Equilibrium (HWE) using Pearson´s χ 2 test. The distribution of genotypes among cases and controls was evaluated by logistic regression analysis. ORs and 95% CI were calculated using STATA v9.2 software (Stata, StataCorp LP, Texas, USA). In order to reduce the effect of confounding factors, corresponding controls for each case group were selected randomly from the whole group using age, gender and ethnicity as matching criteria. In the same study group as presented here, carriers of the sickle cell hemoglobin (HbS) were found to be protected from all major forms of severe malaria, whereas the protection for carriers of the hemoglobin C (HbC) variant was restricted to cerebral malaria. [START_REF] May | Hemoglobin variants and disease manifestations in severe falciparum malaria[END_REF] That study also revealed a lower risk to develop severe malarial anemia in patients with α + -thalassemia. Hence, HbS genotypes were used as a covariate for all comparisons, and genotypes underlying α +thalassemia and HbC were used exclusively in calculations including severe malarial anemia and cerebral malaria phenotypes, respectively. According to the Bonferroni correction for multiple comparisons [START_REF] Bonferroni | Il calcolo delle assicurazioni su gruppi di teste[END_REF], p-values were adjusted as follows: The working hypothesis was that severe malaria may be associated with FCGR2A variants at any mode of inheritance (additive, recessive or dominant) which required a correction factor of 3 for multiple testing.

Then, three phenotypes of severe malaria (severe anemia, cerebral malaria, other manifestations) were tested assuming any mode of inheritance, which required a correction factor of 9. Statistical power to detect a genetic effect in our study group was estimated using CaTS power calculator for association studies. [START_REF] Skol | Joint analysis is more efficient than replication-based analysis for two-stage genome-wide association studies[END_REF] For this purpose a disease prevalence of 2% for severe malaria and a required significance level of 0.005 (correction factor 9) were applied.

RESULTS

The study group consisted of 2504 children with severe falciparum malaria and 2027 apparently healthy controls matched for age, gender and ethnicity (Table 1). 2). In contrast, both FcγRIIa 131HR and FcγRIIa 131HH showed the opposite distribution, suggesting a recessive influence of the FcγRIIa 131R allele. Accordingly, statistical calculations were made under the assumption of a recessive mode of inheritance.

The case group was stratified for the phenotypes of cerebral malaria (n=562) and severe anemia (n=1591), whereby patients with both forms were assigned to each of the two subgroups and those with neither of the two forms were combined in a third subgroup (n=497). The latter included patients with partly overlapping hyperlactatemia (100%), prostration (83%), hyperparasitemia (56%), acidosis (54%), and respiratory distress (27%).

A significant positive association of FcγRIIa 131RR was observed in the subgroup of patients with severe anemia, whereas the frequencies of FcγRIIa 131HR and FcγRIIa 131HH were similar in the two other subgroups with odds ratios (OR) being close to 1 (Table 2). The skewed distribution of genotypes in the severe-anemia subgroup may explain the deviation of allele frequencies from HWE observed in this subgroup.

Given the disease-associated allele frequency of 0.6 in the controls and the estimated OR of 1.33 in the severe anemia subgroup, the statistical power to detect a recessive effect in the phenotypic subgroups is 68% for severe anemia, 20% for cerebral malaria, and 17% in other severe malaria cases. However, when combining all non-anemic severe malaria cases (n=913) and using the complete control group (n=2027), the power achieved is 56%.

Analyzing the genotype distribution among these cases and all available controls revealed no evidence for an effect of FcγRIIa 131RR on the susceptibility to non-anemic severe malaria in our study group (OR, 1.07; CI, 0.91-1.26; p=0.395).

When comparing parasite densities (log-transformed number of asexual parasites per microliter blood) in the genetic subgroups, no significant differences were observed between patients with FcγRIIa 131RR (median, 4.83; 95% confidence interval [CI], 4.76-4.90), FcγRIIa 131HR (median, 4.95; CI, 4.87-5.02), and FcγRIIa 131HH (median, 4.77; CI, 4.63-4.90).

Studying in the same case/control group the association of α + -thalassemia, we had previously noted a difference between patients with severe anemia alone and those with severe anemia plus additional complications. [START_REF] May | Hemoglobin variants and disease manifestations in severe falciparum malaria[END_REF] We had speculated that the two groups might differ in the speed with which anemia develops. Regarding the positive association of severe anemia with FcγRIIa 131RR , we found a discrete but not significant difference between the two subgroups (anemia-alone subgroup, OR, 1. HbS genotypes were used as covariates in all logistic regression analysis. Genotypes for α + -thalassemia and HbC were used exclusively in logistic regressions including severe malarial anemia and cerebral malaria phenotypes, respectively.

a Severe malarial anemia with or without additional complications including cerebral malaria b Cerebral malaria with or without additional complications including severe anemia c Severe malaria complications not including severe anemia or cerebral malaria (hyperlactatemia, 100%; prostration, 83%; hyperparasitemia, 56%; acidosis, 54%; respiratory distress, 27%) d P value corrected by a factor of 3 for multiple testing (3 genetic models): P c3 = .021 e P value corrected by a factor of 9 for multiple testing (3 manifestations in 3 genetic models): P c9 = .009 f Odds ratio g Confidence interval

DISCUSSION

Here we report that FcγRIIa 131RR was positively associated with severe falciparum malaria and, more specifically, with severe malarial anemia, one of the most prevalent malaria complications. No evidence was obtained for any influence of the FcγRIIa H131R polymorphism on cerebral malaria or other major malaria complications in the absence of severe anemia.

The effect of FcγRIIa 131RR on severe malarial anemia appeared to be recessive, as FcγRIIa 131HH and FcγRIIa 131HR showed similar ORs. These findings are only partly in agreement with previous observations. For example, in the largest study on this subject so far, Cooke and co-workers obtained a strikingly different result. [START_REF] Cooke | Association of Fcgamma receptor IIa (CD32) polymorphism with severe malaria in West Africa[END_REF] Including approximately 500 case/controls from The Gambia, they found FcγRIIa 131HH associated with susceptibility to severe malaria including both patients with severe anemia and cerebral malaria. Similarly, two studies on FcγRIIa polymorphisms in study cohorts from Kenya comprising 182 and 493 participants, respectively, provided evidence for protection against malaria by FcγRIIa 131RR , in these cases not against severe disease but against high parasite densities. [START_REF] Shi | Association of FCgamma receptor IIA (CD32) polymorphism with malarial anemia and high-density parasitemia in infants and young children[END_REF]18] On the other hand, one study from India with a total of 1871 individuals and a second study from Sudan investigating 256 individuals showed similar findings as ours but in differently defined malaria phenotypes and without discriminating between the various forms of disease. [START_REF] Nasr | Fc gamma receptor IIa (CD32) polymorphism and antibody responses to asexual blood-stage antigens of Plasmodium falciparum malaria in Sudanese patients[END_REF][START_REF] Sinha | Polymorphisms of TNF-enhancer and gene for FcgammaRIIa correlate with the severity of falciparum malaria in the ethnically diverse Indian population[END_REF] One possible explanation for these discrepancies may be different genetic backgrounds of the ethnically diverse study groups. For instance, linkage disequilibrium has been described between alleles of FCGR2A and FCGR3B, both of which appear to be involved in similar functions and, therefore, may interfere with each other´s evolutionary selection. [START_REF] Van Schie | Evaluation of human FcgammaRIIA (CD32) and FcgammaRIIIB (CD16) polymorphisms in Caucasians and African-Americans using salivary DNA[END_REF] Additionally, epistatic effects involving variants located in other genes in the FCGR gene cluster on chromosome 1q23-24 are likely to exist. A structural polymorphism (V158F) in the extracellular domain of FcγRIIIa has been found to be in linkage disequilibrium with the FcγRIIa H131R polymorphism in Caucasian populations as well as in an Afro-American population. [START_REF] Lejeune | Evidence for linkage disequilibrium between Fcgamma RIIIa-V158F and Fcgamma RIIa-H131R polymorphisms in white patients, and for an Fcgamma RIIIa-restricted influence on the response to therapeutic antibodies[END_REF] A study performed by van der Pol and colleagues underscores the apparent differences in FCGR heterogeneity between ethnic groups, which might explain inconsistent results between different study cohorts. [START_REF] Van Der Pol | Evidence for non-random distribution of Fcgamma receptor genotype combinations[END_REF] Alternatively, it may be argued that several of the previous reports (i) were based on relatively small study groups providing limited statistical power to reveal significant associations with severe malaria, (ii) showed marginal levels of statistical significance and (iii) included stratifications without correcting for multiple testing. As they also yielded largely inconsistent results, some of those studies leave some doubt as to whether the associations reported may have been fortuitous. The study presented here was based on 5 to 20 times more cases and resulted in clearly significant associations or in ORs close to 1 when significance was missing.

Due to the fact that the severe anemia subgroup comprises a nearly three times greater sample size than the other phenotypic subgroups, there is a decline in statistical power to detect the observed effect from 68% in the severe anemia group to 20% and 17% in the two smaller subgroups. A maximum of 56% statistical power can be achieved by analyzing genotype frequencies of all non-anemic severe malaria cases together with the entire control group. Compared to the severe anemia cases and matched controls this analysis provides only marginally less statistical power, and the absence of association of FcγRIIa 131RR with non-anemic severe malaria cases supports the evidence for patients homozygous for FcγRIIa 131R being specifically more susceptible to severe malarial anemia in our study. Notably, in a population with steady exposure to falciparum malaria, the allele frequency of 0.6 of the SMA-associated allele appears to be relatively high. It is likely that the increased affinity between FcγRIIa 131R and CRP exerts a beneficial role in the defense against bacterial pathogens, as CRP binds to several encapsulated bacteria including Haemophilus influenza and Streptococcus pneumonia. [26, 27] Both pathogens were found to be among the leading causes of childhood bacterial meningitis in southern Ghana. [START_REF] Commey | Bacterial meningitis in children in southern Ghana[END_REF] Thus, the R allele could be subject to counterbalancing positive selection maintaining the malaria associated allele in the population.

The association data reported here appear to indicate that certain humoral defense mechanisms contribute to severe malarial anemia substantially more strongly than to other forms of severe malaria. However, addressing the question of whether the loss of the FcγRIIa 131R function to bind IgG 2 and IgG 3 or the gain of function to bind CRP is involved must be highly speculative because, to our knowledge, neither the role of antibody isotypes nor that of CRP has so far been the subject of functional studies in malaria pathology.

If the crucial property of FcγRIIa 131R compared to FcγRIIa 131H would be the reduced ability to mediate the clearance of IgG 2 -and IgG 3 -opsonized parasites or parasitized erythrocytes, patients homozygous for FcγRIIa 131R would be expected to have higher parasite densities than those with FcγRIIa 131HH and FcγRIIa 131HR , which was not the case in our study. In addition, it is difficult to envisage that a reduction in parasite clearance would selectively affect severe anemia rather than all forms of complicated malaria because severe anemia has been found not to be associated with higher parasite densities than coma or other malaria complications. [START_REF] Schofield | Immunological processes in malaria pathogenesis[END_REF] Therefore, it appears unlikely that the loss of binding activity of FcγRIIa 131R for IgG 2 and IgG 3 causes the increase in risk for severe anemia. Rather, the gain of function of FcγRIIa 131R to bind CRP might be relevant. CRP is known to bind to the surface of apoptotic cells [START_REF] Marnell | C-reactive protein: ligands, receptors and role in inflammation[END_REF], and circumstantial evidence suggests that, in severe malarial anemia, nonparasitized erythrocytes acquire surface properties of apoptotic cells.[31] Hence, CRP/FcγRIIa-mediated elimination may contribute to a loss of nonparasitized erythrocytes in severe malarial anemia.

It needs to be stressed that genetic associations as the one presented here provide circumstantial evidence only and do not monitor biological processes directly. They may, however, stimulate further attempts to design experimental and clinical studies to confirm the genetic results at the functional level.

FUNDING

The study was supported by the German National Genome Research Network (NGFN). The NGFN was not involved in any of the following: study design; collection, analysis and interpretation of the data; writing the report; and in the decision to submit the paper for publication.

infants against high-density Plasmodium falciparum infection. VII. Asembo Bay Cohort Project. J Infect Dis 2001;184(1):107-11.

  The non-synonymous substitution of an A by a G in the FCGR2A gene (rs1801274) results in the amino acid substitution at position 131 (H131R) in the second extracellular Iglike domain of the FcγRIIa protein. In this study, the single-nucleotide polymorphism rs1801274 was analyzed by dynamic allele-specific hybridization with fluorescence resonance energy transfer (FRET) in a LightTyper device (Roche Diagnostics, Mannheim, Germany). A fragment of 558 bp of the human FCGR2A gene, which includes the single base polymorphism at position 497 in the coding sequence (c.497A>G), was amplified by PCR. The reaction mixture contained 1x PCR Buffer BD, 1x Solution S, 1 U FIREPol DNA polymerase I, 1.5 mM MgCl 2 (Solis BioDyne, Estonia), 200 µM of each dNTP, 1 µM PCR Primer FCGR2A_for, 0.2 µM of PCR Primer FCGR2A_rev, 0.2 µM FCGR2A_anchor, 0.2 µM FCGR2A_sensor, 2 µl of the WGA sample (1:200 dilution), and water to a final volume of 10 µl. The following reaction conditions were used: 94°C for 3 min, 55 cycles of 94°C for 20 s, 55°C for 30 s, and 72°C for 30 s followed by one cycle of 72°C for 5 min. Primer and sensor/anchor sequences were 5´-CTTATCATATTTRTGTCTTTCAGAATG-3´ (FCGR2A_for), 5´-CAGCCTGACTACCTATTACCTGG-3´ (FCGR2A_rev), 5´-TGCTTGTGGGATGGAGAAGGTGGG-Fluorescein (FCGR2A_anchor), 5´-CY5-TCCAAACGGGAGAATTTCTGGGA-Phosphate-3´ (FCGR2A_sensor). To confirm the validity of the typing method, PCR products of 23 DNA samples were sequenced using primers FCGR2A_ex4-F (5´-TTCTGCTTTTAGAAGTCCCAYAG-3´) and FCGR2A_ex4-R (5´-CAATACTTAGCCAGGCTTCCAC-3´). DNA re-sequencing confirmed the results obtained by FRET genotyping in all cases.

Table 1 .

 1 Demographic Data of the Study Group FCGR2A variants showed that the allele frequencies deviate from HWE exclusively in the group of severe-anemia cases (χ 2 =6.92, p=0.009), but not in the control group and the other subgroups investigated (Supplementary Table1).

			Severe Malaria		Controls
		All severe	Severe Anemia a	Cerebral Malaria b	Others c
		malaria cases
	n	2504	1591	562	497	2027
	Median age (months, range)	20 (7-117)	16 (7-114)	25 (7-117)	27 (7-117)	30 (7-117)
	Male-female ratio	54:46	54:46	54:46	55:45	53:47
	Ethnic group, %
	Akan	67.3	69.2	63.7	64.8	69.2
	Northerners d	29.8	27.8	34.0	31.6	29.3
	Ewe	2.9	3.0	2.3	3.6	1.5

a Severe malarial anemia with or without additional complications including cerebral malaria.

b Cerebral malaria with or without additional complications including severe anemia. c Severe malaria complications not including severe anemia or cerebral malaria (hyperlactatemia, 100%; prostration, 83%; hyperparasitemia, 56%; acidosis, 54%; respiratory distress, 27%).

d

The group of Northeners comprises members of several ethnicities of Northern Ghana Genotyping for

Table 2 .

 2 FcγRIIa H131R Associations in Severe Falciparum Malaria Collectively and Stratified for Major Forms of the Disease

	30; CI, 0.91-1.87; p=0.152; n=261;

Ekvall H. Malaria and anemia. Curr Opin Hematol 2003;10(2):108-14.

ACKNOWLEDGEMENTS

We thank the participating children and their parents and guardians. Tsiri Agbenyega represents the Kumasi team of the Severe Malaria in African Children (SMAC) network, which includes Daniel Ansong, MD, Sampson Antwi, MD, Emanuel Asafo-Adjei, MD, Samuel Blay Nguah, MD, Kingsley Osei Kwakye, MD, Alex Osei Yaw Akoto, MD, and Justice Sylverken, MD, all Komfo Anokye Teaching Hospital, Kumasi.

CONTRIBUTIONS

CONFLICT-OF-INTEREST DISCLOSURE

The authors have no competing interests to declare.

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Boyan Ivanov Dimitrov

Thierry Voet

Joris Luc De Smet

Robert Vermeesch

Koen Devriendt

Jean-Pierre Fryns

Prof. Dr Philippe Debeer
email: philippe.debeer@uzleuven.be

Joris Robert Vermeesch

Genomic rearrangements of the GREM1-FMN1 locus cause Oligosyndactyly, Radio-Ulnar synostosis, Hearing loss, Renal defects syndrome and Cenani-Lenz-like non-syndromic Oligosyndactyly

Keywords: Gremlin1, Formin1, radio-ulnar synostosis, Cenani-Lenz oligosyndactyly, SHFM, limb development

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Vertebrate limb development serves as an important model to show how different processes and pathways are synchronized and involved in morpho-and organogenesis. As limb formation is not necessary for embryonic survival, the study of several spontaneous and molecularly manipulated (transgenic) animal models contributed to the discovery of the many genes involved in limb and skeleton patterning and development. Since several of these key genes play a role in other developmental cascades, it was also believed that this knowledge could be applied to understand the pattern formation of other tissues and organs [START_REF] Niswander | Pattern formation: old models out on a limb[END_REF][START_REF] Tickle | Making digit patterns in the vertebrate limb[END_REF].

The crosstalk between two important signalling centres was found to be crucial in the complex process of limb formation -the Zone of Polarizing Activity (ZPA) and the Apical Ectodermal Ridge (AER). Key components of this mesenchymal-ectodermal (m-e) interaction are Sonic hedgehog (Shh), limb specific Fibroblast Growth Factors (FGFs) , Bone Morphogenic Proteins (BMPs) and BMP antagonists like Gremlin 1 (Grem1). The study of targeted mutations of limb specific FGF and BMP/ BMP antagonist genes revealed the existence of self regulatory loops controlling the appropriate initiation and termination of m-e signalling in the coordination of limb development [START_REF] Zuniga | Signal relay by BMP antagonism controls the SHH/FGF4 feedback loop in vertebrate limb buds[END_REF][START_REF] Khokha | Gremlin is the BMP antagonist required for maintenance of Shh and Fgf signals during limb patterning[END_REF][START_REF] Scherz | The limb bud Shh-Fgf feedback loop is terminated by expansion of former ZPA cells[END_REF][START_REF] Mariani | Genetic evidence that FGFs have an instructive role in limb proximal-distal patterning[END_REF][START_REF] Verheyden | An Fgf/Gremlin inhibitory feedback loop triggers termination of limb bud outgrowth[END_REF][START_REF] Bénazet | A self-regulatory system of interlinked signaling feedback loops controls mouse limb patterning[END_REF]. In addition, the correct spatio-temporal expression of these limb morphogens depends on regulatory sequences surrounding the gene landscape in the vicinity [START_REF] Zuniga | Mouse limb deformity mutations disrupt a global control region within the large regulatory landscape required for Gremlin expression[END_REF][START_REF] Zeller | Shh and Gremlin1 chromosomal landscapes in development and disease[END_REF]. A good example is the limb deformity (ld) mouse. The ld phenotype is autosomal recessively inherited and characterized by distinct reduction defects of the digit rays, complete radioulnar synostosis, missing fibulae and variable renal defects [START_REF] Kleinebrecht | The mouse mutant limb-deformity (ld)[END_REF]. These abnormalities are most likely a result of disturbed function of Grem1 protein in the developing limbs due to homozygous Grem1 mutations or loss/rearrangements of a limb bud specific Grem1 transcriptional global controlling region (GCR) within Fmn1 gene [START_REF] Zuniga | Mouse limb deformity mutations disrupt a global control region within the large regulatory landscape required for Gremlin expression[END_REF][START_REF] Michos | Gremlinmediated BMP antagonism induces the epithelial-mesenchymal feedback signaling controlling metanephric kidney and limb organogenesis[END_REF][START_REF] Pavel | Analysis of a new allele of limb deformity (ld) reveals tissue-and age-specific transcriptional effects of the Ld Global Control Region[END_REF]. The importance of Grem1 in limb development is further supported in that its over-expression in chick limb primordia results in truncations of distal autopod elements and syndactyly [START_REF] Capdevila | Control of vertebrate limb outgrowth by the proximal factor Meis2 and distal antagonism of BMPs by Gremlin[END_REF][START_REF] Merino | The BMP antagonist Gremlin regulates outgrowth, chondrogenesis and programmed cell death in the developing limb[END_REF]. Recent studies reinforce the hypothesis that a disturbed Fmn1 protein function in limb buds may also contribute to the abnormal mouse ld phenotype [START_REF] Zhou | Formin1 disruption confers oligodactylism and alters Bmp signaling[END_REF].

Given the recent observation that causal submicroscopic copy number variations (CNV's) can be detected in a significant proportion of patients with unexplained congenital malformations, we analyzed seventeen individuals with either non-classified syndromic congenital skeletal anomalies or severe classified limb malformations of unknown molecular aetiology. The study was approved by the Ethics Committee of the University Hospitals Leuven. With a homemade 1Mb array Comparative Genomic Hybridization (CGH) (Supplemental data), two patients with a chromosomal rearrangement of the GREM1-FMN1 locus on chromosome 15q13.3 were identified. In the first patient (C255625) with oligosyndactyly of the four limbs, radioulnar synostosis, hearing loss and unilateral renal aplasia (Figure 1a-d), a homozygous deletion of one BAC clone (RP11-250G15) was detected (not shown). He was previously reported as a case of Split-Hand-Foot-Malformation 1 (SHFM1) [START_REF] Debeer | Bilateral complete radioulnar synostosis associated with ectrodactyly and sensorineural hearing loss: a variant of SHFM1[END_REF]. In the second patient (C80286), a two clone duplication (RP11-3D4 and RP11-250G15) encompassing the GREM1-FMN1 locus was detected (not shown). This individual had nonsyndromic Cenani-Lenz type oligosyndactyly (Figure 1e-g) [START_REF] De Smet | Cenani-Lenz type of syndactyly: a complex type of syndactyly with multiple synostoses[END_REF]. No aberrations were detected in the remaining fifteen patients. For the phenotypes of the analyzed individuals and detailed reports of Patient 1 and Patient 2 with the detected microdeletion/duplication, see the Supplemental data and Supplemental table 1.

Subsequent FISH analysis (Supplemental data) using probe RP11-250G15 (red signal) confirmed the presence of a homozygous deletion in patient C255625 (Figure 2a). Several unaffected individuals from the same family were carriers of a heterozygous deletion (Figure 2a). FISH screening with probes RP11-250G15 (red signal) and RP11-3D4 (green signal) showed the de novo 15q13.3 tandem duplication in patient C80286 (Figure 2b).

The micro-deletion/duplication were further delineated by high resolution SNP array (Affymetrix 250K NspI, Supplemental data) in patient C255625, his parents (C359651 and C359656) and case C80286. The homozygous deletion size was maximally 263kb (between SNPs SNP_A-4201883 and SNP_A-2233558) and minimally 246kb (between SNPs SNP_A-4240484 and SNP_A-2176534), encompassing the first twelve FMN1 exons and non-coding upstream sequences (Figure 3a-c). The healthy parents (C359651 and C359656) were heterozygous carriers of the same aberration (not shown). The duplication in patient C80286 had a maximum size of 2.3Mb (between SNP_A-2022976 and SNP_A-4217332) and a minimum size of 1.7Mb (between SNP_A-2284222 and SNP_A-2248182), including both GREM1 and FMN1 (Figure 3a-c). Other previously undetected CNVs, that could possibly explain the phenotype of these two affected individuals, were also excluded with the SNP array.

The quality of the SNP array CGH results was validated by RT Q-PCR (Supplemental data).

Twelve primer pairs, overlapping or in the vicinity of the detected deletion/duplication breakpoints, were designed. All results were in concordance with the SNP array CGH data (not shown). One assay was used to demonstrate the de novo character of the microduplication in patient C80286, (Figure 3d).

To investigate the effect of these rearrangements on GREM1 and FMN1 expression, we studied the gene expression in the fibroblasts of both patients (Supplemental data). As a highly variable expression pattern was observed in normal controls, a significant quantitative analysis was not possible (not shown). Of interest, in the patient C255625 with a homozygous deletion of the promoter and the first twelve FMN1 exons, a low expression of the FMN1 gene was detected (not shown).

The importance of the GREM1-FMN1 locus in limb development has been extensively studied in animal models. We present for the first time evidence that this locus is implicated in human limb development. In one patient [START_REF] Debeer | Bilateral complete radioulnar synostosis associated with ectrodactyly and sensorineural hearing loss: a variant of SHFM1[END_REF] a homozygous deletion of the first twelve 5' exons of the FMN1 gene was detected. His phenotype is highly reminiscent of the ld mice with oligosyndactyly, radio-ulnar synostosis and renal aplasia (Figure 1a-d). The phenotypic abnormalities in this individual are similar to those of animal models in which the function of Grem1 was abrogated, thus resulting in an abnormal m-e interaction during limb and metanephric kidney organogenesis [START_REF] Khokha | Gremlin is the BMP antagonist required for maintenance of Shh and Fgf signals during limb patterning[END_REF][START_REF] Scherz | The limb bud Shh-Fgf feedback loop is terminated by expansion of former ZPA cells[END_REF][START_REF] Verheyden | An Fgf/Gremlin inhibitory feedback loop triggers termination of limb bud outgrowth[END_REF][START_REF] Bénazet | A self-regulatory system of interlinked signaling feedback loops controls mouse limb patterning[END_REF][START_REF] Zuniga | Mouse limb deformity mutations disrupt a global control region within the large regulatory landscape required for Gremlin expression[END_REF][START_REF] Michos | Gremlinmediated BMP antagonism induces the epithelial-mesenchymal feedback signaling controlling metanephric kidney and limb organogenesis[END_REF][START_REF] Pavel | Analysis of a new allele of limb deformity (ld) reveals tissue-and age-specific transcriptional effects of the Ld Global Control Region[END_REF]. In addition he has hearing loss, which has not yet been observed in the animal model. The second patient carried a de novo 1.7 Mb duplication encompassing the GREM1-FMN1 locus. His isolated Cenani-Lenz-like oligosyndactyly phenotype (Figure 1e-g) [START_REF] De Smet | Cenani-Lenz type of syndactyly: a complex type of syndactyly with multiple synostoses[END_REF] is similar to that of the transgenic chick in which Grem1 over-expression causes primordial cartilage truncations and digit syndactyly due to a repression of the BMP dependant programmed cell death in the anterior necrotic zone and in the inter-digital mesenchyme. The result is a completely disorganized bone pattern of the distal autopod [START_REF] Capdevila | Control of vertebrate limb outgrowth by the proximal factor Meis2 and distal antagonism of BMPs by Gremlin[END_REF][START_REF] Merino | The BMP antagonist Gremlin regulates outgrowth, chondrogenesis and programmed cell death in the developing limb[END_REF].

The phenotype in ld mice is due to a disturbed function of the BMP antagonist Grem1 either by homozygous Grem1 mutations or a loss of Grem1 transcriptional GCR located within the neighbouring Fmn1 gene (Figure 4) [START_REF] Khokha | Gremlin is the BMP antagonist required for maintenance of Shh and Fgf signals during limb patterning[END_REF][START_REF] Zuniga | Mouse limb deformity mutations disrupt a global control region within the large regulatory landscape required for Gremlin expression[END_REF][START_REF] Michos | Gremlinmediated BMP antagonism induces the epithelial-mesenchymal feedback signaling controlling metanephric kidney and limb organogenesis[END_REF][START_REF] Pavel | Analysis of a new allele of limb deformity (ld) reveals tissue-and age-specific transcriptional effects of the Ld Global Control Region[END_REF]. Therefore, the coding part of GREM1 gene was directly sequenced in the fifteen patients in whom no causal CNVs were detected by the array CGH screening (Supplemental data). No mutations were found. In mice, the genomic region between the nineteenth and twenty-third Fmn1 exons contains regulatory sequences controlling the Grem1 expression in developing limbs and kidneys (Figure 4b) [START_REF] Zuniga | Mouse limb deformity mutations disrupt a global control region within the large regulatory landscape required for Gremlin expression[END_REF]. We determined the human homologue of this region using BLAST (blastn and bl2seq) and VISTA (genomeVISTA) alignments [START_REF] Mcginnis | BLAST: at the core of a powerful and diverse set of sequence analysis tools[END_REF][START_REF] Frazer | VISTA: computational tools for comparative genomics[END_REF]. The familial deletion of the first twelve FMN1 exons observed in patient C255652 does not remove these regulatory sequences (Figure 4a,b). Therefore they were not included in the mutational analysis. Point mutations or undetected submicroscopic chromosomal aberrations of these or other limb specific GREM1 regulatory sequences cannot be excluded in the fifteen individuals with negative mutation and CNV screening. There could also be a patient selection bias, since ld mice carrying Grem1 mutations have a more severe and sub-lethal phenotype [START_REF] Michos | Gremlinmediated BMP antagonism induces the epithelial-mesenchymal feedback signaling controlling metanephric kidney and limb organogenesis[END_REF]. Hence, the corresponding human phenotype could be different to those of our selected group [START_REF] Kleinjan | Long-range gene control and genetic disease[END_REF].

In addition, the phenotype of the recently published ld strain is a result of a targeted deletion of Fmn1 exon nine. This leads to a complete loss of any Fmn1 transcript, an unexpectedly extended zone of Grem1 expression and an increased Bmp activity in ld limb buds, indicating a direct Fmn1 repression of the Bmp signalling. However the authors do not exclude the possibility that a loss of regulatory sequences could also explain the phenotype of their animal model [START_REF] Zhou | Formin1 disruption confers oligodactylism and alters Bmp signaling[END_REF]. Using computer analysis we searched for the presence of evolutionarily conserved sequences in the deleted genomic fragment in our patient. We found a few non-coding elements that could have a regulatory function and which were highly homologous through all tetrapods and chick but not present in xenopus, zebrafish and fugu [START_REF] Loots | Genomic identification of regulatory elements by evolutionary sequence comparison and functional analysis[END_REF]. Unfortunately, a direct proof of causal relationship between tissue specific regulatory sequences and their corresponding genes in humans is limited due to restrictions in obtaining appropriate patient samples for analysis (i.e. tissues in which the gene of interest is expressed at specific time points). All these findings make it difficult to judge to what extent either GREM1 or FMN1 contribute to the phenotype of the carrier of the homozygous 15q13.3 deletion in Family 1.

High resolution SNP array analysis of the breakpoints of the second patient (C80286) showed that low copy repeats (LCR) BP5 and BP6 on chromosome 15q13.3q14.1 flank the detected duplication. Thus the aberration is proximal to the newly recognized microdeletion 15q13.3 syndrome defined between LCR blocks BP3-BP4 and BP5 [START_REF] Sharp | A recurrent 15q13.3 microdeletion syndrome associated with mental retardation and seizures[END_REF]. Previous studies focused on the analysis of the 15q13.3 region did not identify a similar BP5-BP6 duplication in 3699 North American controls of predominantly European ancestry [START_REF] Helbig | 15q13.3 microdeletions increase risk of idiopathic generalized epilepsy[END_REF]. Analysis of 2590 individuals in our Centre by 1Mb array CGH revealed a maternally inherited benign hemizygous deletion reciprocal to the duplication in patient C80286. Hence, there is an equal proportion of detected benign hemizygous deletions and disease causing duplications for the locus between LCR BP5-BP6. None of the few published patients with larger proximal 15q13q14 deletions encompassing the BP5-BP6 locus had distal limb anomalies [START_REF] Matsumura | Severe' Prader-Willi syndrome with a large deletion of chromosome 15 due to an unbalanced t(15,22)(q14;q11.2) translocation[END_REF][START_REF] Windpassinger | Molecular characterization of a unique de novo 15q deletion associated with Prader-Willi syndrome and central visual impairment[END_REF][START_REF] Erdogan | Characterization of a 5.3 Mb deletion in 15q14 by comparative genomic hybridization using a whole genome "tiling path" BAC array in a girl with heart defect, cleft palate, and developmental delay[END_REF].

The duplication of patient C80286 includes at least fourteen other genes besides GREM1 and FMN1 (Figure 3b). Currently there is no evidence in the literature that overexpression of these other genes would contribute to an abnormal limb development in humans or animal models. However, we do believe that the de novo character of the observed duplication in patient C80286, the absence of similar chromosomal rearrangements in a large cohort of controls screened in previous surveys [START_REF] Sharp | A recurrent 15q13.3 microdeletion syndrome associated with mental retardation and seizures[END_REF][START_REF] Helbig | 15q13.3 microdeletions increase risk of idiopathic generalized epilepsy[END_REF] and the strong support of an existing corresponding animal model with similar skeletal defects [START_REF] Capdevila | Control of vertebrate limb outgrowth by the proximal factor Meis2 and distal antagonism of BMPs by Gremlin[END_REF][START_REF] Merino | The BMP antagonist Gremlin regulates outgrowth, chondrogenesis and programmed cell death in the developing limb[END_REF] confirm the causal relationship between the patient's phenotype and the increased copy number at the GREM1-FMN1 locus. Since LCRs flank the detected aberration (Figure 3b), the most probable causative mechanism is non-allelic homologous recombination (NAHR) [START_REF] Gu | Mechanisms for human genomic rearrangements[END_REF].

In summary, the present study delineates for the first time the human phenotype associated with a homozygous microdeletion of the FMN1 gene. The patient's skeletal and renal defects are identical to those of ld mice. We also detected the human phenotype associated with a microduplication of the GREM1-FMN1 locus in an individual with bilateral hand oligosyndactyly. No mutations of the GREM1 gene were found among individuals with similar limb defects and/or renal anomalies. The low yield of our screening to detect genomic aberrations/point mutations in additional cases may be explained by a patient selection bias or due to restriction of the mutation analysis to the coding sequence of GREM1. Cenani-Lenz syndrome (CLS) was defined as a form of hand and foot syndactyly due to complete cutaneous and bony fusions. This results in disorganisation and sometimes loss of individual digit rays, and spoon-like hands. The feet are similarly affected but to a less degree.

Radioulnar synostosis, mesomelic shortness, renal hypoplasia and other miscellaneous anomalies have been also observed [START_REF] Harpf | A variant of Cenani-Lenz syndactyly (CLS): review of the literature and attempt of classification[END_REF]. No linkage to the GREM1-FMN1 locus was found in a consanguineous family with offspring with classical spoon-like hands/ renal hypoplasia CLS [START_REF] Bacchelli | Cenani-Lenz syndrome with renal hypoplasia is not linked to FORMIN or GREMLIN[END_REF]. Hence, even though our patients share some common clinical features [START_REF] Frazer | VISTA: computational tools for comparative genomics[END_REF]. There is bilateral oligosyndactyly with four fingers on the right hand (1a) and three on the left (1b). The nails of the digits are normal.

Both feet have an absent second ray and four remaining toes. (1c). An excretory contrast Xray examination demonstrates agenesis of the left kidney (1d). The phenotype of patient C80286 was previously classified as an isolated Cenani-Lenz-like oligosyndactyly of all fingers of both hands [START_REF] Kleinjan | Long-range gene control and genetic disease[END_REF]. Radiographs of the hands at 6 months of age (1e) and at 27 years of age (1f) show progressive multiple fusions of metacarpal bones and phalanges with several missing skeletal elements. Fusion of carpal bones is also present. Both feet have no skeletal anomalies (1g). Appropriate informed consent was obtained from the participating patients or their legal representatives. . This is in correlation with the array CGH results and demonstrates that the patient has a homozygous deletion of the genomic locus covered by this probe. FISH analysis showed a hybridization of BAC RP11-250G15 (red signal) on only one metaphase chromosome fifteen in heterozygous carriers of the aberration in Family 1, the parents (C359656 and C359651) and two additional siblings (C367866 and C367867) respectively. In addition, a cross-hybridization of BAC RP11-250G15 (red signal) on chromosome 3q was also observed in all analyzed metaphases. B) To confirm the presence of the microduplication in patient C80286, FISH analysis was done on interphase nuclei using two FISH probes residing within the aberration, BAC RP11-250G15 (red signal) and BAC RP11-3D4 (green signal). All patient's nuclei had one pair of a red and a green signal representing the normal allele, and a string of two red and two green signals corresponding to the allele carrying the duplication. Arrow points the aberrant allele (with duplication). The linear order of these four signals was always red-green-red-green suggesting a tandem orientation of the duplicated fragments. Both parents (C80284 and C80285) were not carriers of the aberration. In addition, all analyzed patient's and parental nuclei presented two more red signals. These two additional signals were every time separated from the others and were not paired with green one, demonstrating a cross-hybridization of BAC RP11-250G15 on chromosome 3q. Subsequently, this cross-hybridization was also confirmed on parental and patient's chromosomal metaphase spreads (not shown).

Appropriate informed consent was obtained from the participating patients or their legal representatives.

(

  oligosyndactyly, radioulnar synostosis and renal abnormalities) with published CLS cases, they represent unique phenotypes caused by GREM1/FMN1 alterations. Of course, genetic heterogeneity could be expected since abnormalities of genes participating or interacting with the SHH-GREM1-FMN1-FGF8/4 pathway in developing limbs, might cause identical/similar developmental defects. Therefore, our patients represent new clinical entities within the Cenani-Lenz phenotypic spectrum-(1) an autosomal recessive Oligosyndactyly, Radio-Ulnar Synostosis, Hearing loss and Renal defect syndrome due to a homozygous genomic deletion of FMN1 gene, and (2) a dominant Cenani-Lenz-like non-syndromic oligosyndactyly caused by a duplication of GREM1-FMN1 locus. The detected tandem 15q13.3q14 duplication delineates a new candidate for recurrent genomic disorder [33].Analyzing more patients and further research will be necessary to unravel the clinical extent of these two new phenotypes/syndromes.

Figure 1 .

 1 Figure 1. Clinical phenotype of patients C255625 and C80286. Patient C255625 was published as an atypical case of SHFM1[START_REF] Frazer | VISTA: computational tools for comparative genomics[END_REF]. There is bilateral oligosyndactyly with four

Figure 2 .

 2 Figure 2. Pedigrees of patient C255625 (Family 1) and patient C80286 (Family 2). Below each individual partial metaphase spreads or interphase nuclei are shown summarizing the results of the performed FISH analysis. Filed red and green colour squares represent the patients. Striped red circles and squares show heterozygous carriers in Family 1. A) BAC RP11-250G15 (red signal), with genomic position as shown on Figure 3c, was used as a FISH probe to confirm the abnormalities detected by the array CGH screening in Family 1. A chromosome 15q subtelomeric probe (green signal) served as a control. Arrows point to the aberrant alleles (with deletion) in the analyzed individuals. There was no hybridization of BAC RP11-250G15 (red signal) on both chromosomes 15 in patient C255625. This is in

Figure 3 .

 3 Figure 3. GREM1-FMN1 locus on chromosome 15q13.3 and genomic positions of the detected homozygous deletion (C255625) and de novo duplication (C80286) according the Human Reference Sequence March 2006, NCBI Build 36.1. A) Fine mapping of the breakpoints by 250 NspI SNP array of Affymetrix in patient C255625 and C80286. B) Genomic architecture of the aberrant locus. Transparent vertical green and pink lines demarcate the positions of the breakpoints. Low copy repeats (BP5 and BP6) flank the detected de novo 1.7Mb (minimal size) tandem duplication (dark green bar) of patient C80286. C) Colour bars and triangles show the positions of the used BACs as FISH probes and some of the RT Q-PCR primers. Dark red and green bars represent the homozygous deletion (minimal size) of C255625 and the tandem de novo duplication (minimal size) of C80286, respectively. D) Results of the RT Q-PCR with five of the primers used to confirm the SNP array CGH results and their corresponding genomic locations according the detected chromosomal aberrations in Family 1 and Family 2 (Supplemental table 1). The bars visualize the present relative allele copy number (fold differences). Value of 1 stands for the normal diploid status, 0.5 is for hemizigousity, 0 for homozygous deletion and 1.5 for a duplication, respectively. The closest outside primers flanking the breakpoints of the detected homozygous deletion in case C255625 (Family 1) are shown in blue (Pr5) and yellow (Pr10) colours. The red (Pr6) and pink (Pr9) bars represent the closest inside primers to the breakpoints of the same patient. The green bar demonstrates the primer (Pr7) used for detection of the parental origin of the duplication in case C80286 (Family 2). Both parents (C80284 and C80285) have a normal diploid allele copy number. Appropriate informed consent was obtained from the participating patients or their legal representatives.

Figure 4 .

 4 Figure 4. Alignment of the human and mouse GREM1-FMN1 locus. A) The human GREM1-FMN1 locus (Human Reference Sequence March 2006, NCBI Build 36.1) and the detected in Family 1 deletion (transparent pink bar). B) The mouse Grem1 and Fmn1 genomic position

ACKNOWLEDGEMENTS P.D. and K.D. are Clinical Research Investigators of the Fund for Scientific Research, Flanders, Belgium. B.D. was partially supported by grant EO/06/32 of the Catholic University of Leuven, Belgium.

Competing Interest: None to declare.

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