Dr Ruth A Newbury-Ecob
email: ruth.newbury-ecob@uhbristol.nhs.uk.

Jan Maarten Cobben

Gabriele Gillessen-Kaesbach

Judith Goodship

Hanne Hove

Susanne Kjaergaard

Helena Kemp

Helen Kingston

Peter Lunt

Sahar Mansour

Rob Hastings

Jan-Maarten Cobben

Ruth Mcgowan

Kay Metcalfe

Catherine Murdoch- Davis

Mary Ray

Marlène Rio

Sarah Smithson

John Tolmie

Peter Turnpenny

Bregje Van Bon

Dagmar Wieczorek

Bohring -Opitz

Bohring-Opitz (Oberklaid-Danks) syndrome: Clinical study, review of the literature and discussion of possible pathogenesis

Keywords: Bohring-Opitz, Oberklaid-Danks, cholesterol biosynthesis, array CGH, trigonocephaly

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INTRODUCTION

Bohring-Opitz syndrome (BOS) was first described in 1999 by Bohring et al [START_REF] Bohring | Severe end of Opitz trigonocephaly (C) syndrome or new syndrome?[END_REF] , who described four new patients and identified similarities with two patients who had previously been reported as having Opitz C syndrome [START_REF] Oberklaid | The Opitz trigonocephaly syndrome[END_REF][START_REF] Addor | C" trigonocephaly syndrome with diaphragmatic hernia[END_REF] . As one of the patients was initially described by Oberklaid and Danks 2 this syndrome is sometimes known as Oberklaid-Danks syndrome, but we will use the more commonly used attribution of BOS henceforth. To our knowledge a total of twenty-one cases have been reported in the medical literature to date. We propose diagnostic criteria for the clinical diagnosis of BOS, describe ten previously unreported patients and provide an update on four of those previously reported [START_REF] Greenhalgh | Siblings with Bohring-Opitz syndrome[END_REF][START_REF] Bisgaard | Transmitted cytogenetic abnormalities in patients with mental retardation: Pathogenic or normal variants[END_REF][START_REF] Simpson | Infantile high myopia in Bohring-Opitz syndrome[END_REF] . The characteristic features of this condition are typical facial appearance (trigonocephaly/prominent metopic ridge, retrognathia, prominent eyes with hypoplastic supraorbital ridges, upslanting palpebral fissures, depressed nasal bridge, anteverted nares, low-set and posteriorly rotated ears, palatal abnormalities and broad alveolar ridges, flammeus nevus, low anterior hairline), microcephaly, IUGR and short stature, joint abnormalities, abnormal tone, severe/profound developmental delay, susceptibility to infections, feeding difficulties, and high infant mortality. Due to the overlap of many of the clinical features of BOS with some of the disorders of cholesterol biosynthesis, such as Smith-Lemli-Opitz syndrome, desmosterolosis, and lathosterolosis, we decided to analyze cholesterol biosynthesis precursors using tandem mass spectrometry.

MATERIALS AND METHODS

Patients

In this study fourteen patients with the clinical features of BOS have been included.

All patients had a normal karyotype on standard, G-banded, chromosome analysis. Hastings et al As molecular confirmation of the diagnosis is not yet possible and there are no set diagnostic criteria we have included those patients who have at least 7 of the 10 more common and/or specific features. These are: 1) trigonocephaly, 2) primary or secondary microcephaly, 3) flammeus nevus, 4) prominent eyes, 5) micro-or retrognathia, 6) abnormal palate, 7) typical BOS posture*, 8) feeding difficulties, 9) IUGR and 10) Severe/profound learning difficulties or death before this can be adequately assessed.

* BOS-posture of the upper limbs is defined as having 3 out of 4 features: exorotation and/or adduction of the shoulders; flexion at the elbows; flexion at the wrists; ulnar deviation of the wrists and/or fingers at the MCP joints.

Biochemical study: Analysis of the cholesterol biosynthesis pathway

Cholesterol precursors were analyzed as described by Kelley 7 . The precursors were liberated from their fatty acid esters by saponification using methanolic tetramethylammonium hydroxide and extracted into a hydrophobic solvent. Extracts were then dried under nitrogen and converted to heat stable trimethylsilyl (TMS) derivatives using trimethylchorosilane. Analysis was then performed by gas chromatography mass spectrometry which both separated and detected the following cholesterol precursors: 7-dehydrocholesterol, 8-dehydrocholesterol, cholestanol, desmosterol, lathosterol and lanosterol. Individual precursors were identified by monitoring specific ions and quantitation was achieved using an internal standard and calibration material with known amounts of each cholesterol. These studies were performed in patients 1, 2 and 3.

Genetic study: Array comparative genomic hybridization (CGH) Hastings et al

Array CGH was performed using standardized protocols with differing platforms used according to local arrangements (using the Bluegnome cytochip 1 MB BAC array, tiling path 32K BAC array [START_REF] Bisgaard | Transmitted cytogenetic abnormalities in patients with mental retardation: Pathogenic or normal variants[END_REF] , or the Agilent Wessex customized 44K oligoarray). All patients except patients 4 and 12 had array CGH performed.

RESULTS

Clinical data

The clinical details of patients are described below and summarized in table 1. Facial photographs of patients are shown in figure 1 and general photos in figure 2.

Patients 1 and 2 have previously been reported as they are the only siblings reported with clinical features of BOS [START_REF] Greenhalgh | Siblings with Bohring-Opitz syndrome[END_REF] . Patient 1 is a male born to non-consanguineous Caucasian British parents. He was born at thirty-eight weeks gestation by emergency cesarean section for fetal distress. His birth weight was 3.23kg (10 th centile) and OFC 33.8cm (25 th centile). He had trigonocephaly, prominent eyes with upslanting palpebral fissures and hypertelorism, depressed nasal bridge, low-set and dysplastic ears, narrow U-shaped palate with broad alveolar ridges, micrognathia, sacral dimple, and nevi flammei on the philtrum, nape of the neck and forehead. He had truncal hypotonia but limb hypertonia with an unusual posture characterized by the shoulders being held externally rotated and adducted, elbows flexed, wrists flexed and ulnar deviated, and ulnar deviation at the metacarpophalangeal joints (referred to from here on as the 'BOS posture'), and also contractures of the elbows and fingers and gracile ribs. Initially he had respiratory distress and episodes of hypoglycaemia. He had significant feeding difficulties, recurrent vomiting, and failure to thrive requiring gastrostomy feeding and fundoplication surgery. Feeding has improved but he Hastings et al continues to be gastrostomy fed along with some oral intake. At age fourteen he had bowel obstruction secondary to volvulus requiring partial colectomy. In early childhood he had significant problems with recurrent chest and urinary tract infections which have become less frequent. He has a unilateral squint. Weight and head circumference rapidly dropped in infancy to below the 0.4 th centiles but have stabilised from early childhood, at age fifteen his height is 123 cm (<<0.4 th centile), weight 27.0 kg (<0.4 th centile) and OFC 49.5 cm (<<0.4 th centile). He has severe developmental delay but has made slow developmental progress, being able to stand with support and showing reasonable understanding but without meaningful communication. Cranial MRI showed a relatively small brain with expansion of right frontal horn of the lateral ventricle, slender corpus callosum and brainstem, and abnormal neuronal migration with focal subependymal nodular heterotopia.

Patient 2 was born at thirty-seven weeks gestation by emergency cesarean section due to placenta previa. She weighed 2.56kg (25 th centile) and had a head circumference of 32.7cm (25 th centile). The pregnancy was complicated by gestational diabetes. Dysmorphic features were similar to her brother. She had the same 'BOS posture' with fixed contractures at the elbows. Clinical course and developmental progression has been similar but slightly worse than her brother. In early childhood infections were a recurrent problem, particularly chest infections but also pneumococcal meningitis. Problems with feeding and recurrent vomiting have required gastrostomy feeding and fundoplication, vomiting has improved but she remains gastrostomy fed. She has a squint and had delayed visual maturation. Absence seizures in early childhood were treated with levitaracetam. She also had cyanotic/apneic episodes. At age ten she was diagnosed with proximal renal tubular acidosis leading to fragility Dysmorphic features included trigonocephaly with fused metopic suture, prominent eyes, hypertelorism, epicanthal folds, glabella nevus flammeus, depressed nasal bridge, broad alveolar ridges, retrognathia, and low temporal hairline. BOS posture was present along with contractures of fingers, knees, and elbows with limited spontaneous movement. Development was severely delayed. She had feeding difficulties and failure to thrive. She has had generalized seizures and EEG showed hypsarrhythmia. Ophthalmic problems consisted of squint and nystagmus. She also had an umbilical hernia. At fifteen months of age her height was 66cm (<0.4 th centile), weight 7.9kg (<0.4 th centile) and OFC 36cm (<<0.4 th centile). Cranial MRI Hastings et al showed leukomalacia. Recurrent infections were problematic and she died at 2 years of age due to pneumonia.

Patient 10 was born at term to non-consanguineous Turkish parents. Birth weight was 1.8kg (<0.4th centile) and length 42cm (<0.4th centile). Dysmorphic features included prominent eyes, upslanting palpebral fissures, broad alveolar ridges, retrognathia, low temporal hairline, and hypertrichosis. BOS posture was present, the thumbs were adducted and the fingers had contractures as well as elbows and knees. Scoliosis was also present. She had median cleft palate, feeding difficulties requiring tube feeding and failure to thrive. Other problems included recurrent infections and profound sensorineural hearing loss. Ophthalmological examination revealed atrophy of retinal epithelium and nystagmus. Seizures developed in infancy and were treated with valproate. Growth parameters fell further from the normal range and at the age of 5 years she had a height of 90 cm (<0.4 th centile), a weight of 12 kg (<0.4 th centile) and an OFC 44 cm (<<0.4 th centile). Echocardiography showed a secundum atrial septal defect. She died at the age of six years due to pneumonia.

Patient 11 was born by cesarean section at thirty-eight weeks of gestation to nonconsanguineous Dutch Caucasian parents with a birth weight of 2.97kg (25 th centile).

Bilateral cleft lip and clenched hands had been detected antenatally. He had bilateral cleft lip and palate, prominent eyes with hypoplastic supraorbital ridges, hypertelorism, midfacial capillary hemangioma, thick hair with low temporal hairline, and absence of distal interphalangeal creases from the 2 nd -4 th fingers of the right hand and 3 rd and 4 th fingers of the left hand. He had feeding difficulties and failure to thrive. Inguinal hernia was corrected at two and a half months of age. He had high Hastings et al myopia, squint, and high intraocular pressures initially. BOS posture, hypotonia, and bilateral hip dysplasia were present. At eight months of age absence seizures developed with tonic-clonic seizures appearing at one year of age, with epileptiform activity being confirmed on EEG. Head circumference was 43cm at nine and a half months of age (0.4 th centile) and trigonocephaly became evident at one year of age.

Development has been severely delayed, at two and a half years of age he can roll over but cannot sit unsupported and does not speak. His height is 98cm (98 th centile) and weight 14kg (50 th centile). Cranial MRI demonstrated partial agenesis of the corpus callosum.

Patient 12 was born at term to consanguineous Pakistani parents weighing 2.16kg (2 nd centile). He had feeding difficulties and failure to thrive. He had low temporal hairline, synophrys, facial hemangioma, prominent eyes with hypoplastic supraorbital ridges, hypertelorism, short philtrum, tented upper lip, high V-shaped palate with thickened alveolar ridges, micrognathia, hyperconvex nails, BOS posture, and rockerbottom feet. He also had bilateral hypoplastic irides with anterior segment dysgenesis, scleralisation of superior cornea, markedly cupped pale discs, pale retinal pigment epithelium/prominent choroidal vessels, salt and pepper retinal changes, poorly defined macular regions, and high myopia. He developed secondary microcephaly.

Cranial MRI showed agenesis of the corpus callosum and cerebellar hypoplasia.

Echocardiogram showed hypertrophic cardiomyopathy. He died at 13 months from sepsis.

Patient 13 was born at 38 weeks gestation by elective cesarean section to nonconsanguineous Indian parents. Antenatally a nuchal fold measurement of 2.4mm Hastings et al (normal <2mm) and IUGR were noted. He was born weighing 2.3kg (2 nd -9 th centile) and OFC of 33cm (25 th centile). He had a small anterior fontanelle, ridged metopic suture, unilateral cleft lip, broad alveolar ridges, high palate, long uvula, prominent eyes with proptosis and hypertelorism, persistent pupillary membranes, low-set dysplastic ears, micrognathia, short neck, wide spaced nipples, hypotonia, and BOS posture with long clenched fingers and right positional foot deformity but no true contractures initially. At two months of age knee and elbow flexion deformities were more prominent with contractures of the elbows developing. Inguinal hernia was also noted. Feeding difficulties and reflux required nasogastric feeding. Echocardiogram showed a PFO. CT head was normal. Hearing assessment demonstrated no response on the left, normal on the right. He had recurrent chest infections and died of cardiorespiratory problems, preceded by apneic episodes and bradycardias, at six months of age.

Patient 14 has previously been reported to highlight the ophthalmic features of BOS, particularly high myopia [START_REF] Simpson | Infantile high myopia in Bohring-Opitz syndrome[END_REF] . She was born at term to non-consanguineous Caucasian British parents weighing 2.9kg (10 th centile) and OFC was 32cm (2 nd centile).

Antenatally a raised nuchal translucency was noted. She had trigonocephaly, facial flammeus nevus, a capillary hemangioma on the left cheek, low anterior hairline, upslanting palpebral fissures and prominent eyes, depressed nasal bridge and anteverted nares, small mouth, broad alveolar ridges and a narrow high-arched palate with sub-mucous cleft, along with the BOS posture and other joint abnormalities (genu recurvatum, congenital hip dysplasia, and knee and ankle dislocations). She required gastrostomy feeding for feeding difficulties and failure to thrive. She has had frequent respiratory infections and an episode of pneumococcal meningitis. She has had difficulties with upper airway obstruction and has required grommets for moderate conductive hearing loss. In infancy she had focal and generalized seizures but these are well controlled with valproate. She had bilateral femoral fractures at the age of two years due to osteopenia of unknown cause. She has exophthalmos, strabismus, retinal abnormalities, and high myopia. Development has been profoundly delayed and at the age of four-and-a-half years she is unable to sit independently or make any meaningful communication. Her OFC is 46.5cm (<<0.4 th centile).

Analysis of the cholesterol biosynthesis pathway

Compared to normal plasma samples there was no increase in the sterol profile in patients 1, 2 and 3 (other patients were not analysed in this manner). 7-DHC was analysed in patient 9 and was normal.

Array CGH

Patient 7 had a maternally inherited deletion on chromosome 10q11.21-23 detected by metaphase high-resolution CGH. This was also found in two healthy siblings, and was similar to deletions previously reported without a recognizable phenotype, and was therefore felt unlikely to be pathogenic. Patient 10 had a maternally inherited 388kb deletion at 13q33.3 not containing any known OMIM Morbid genes, and again therefore was felt unlikely to be pathogenic.

DISCUSSION

From the published patients and our cohort the consistent clinical features of this rare condition are becoming clearer: Hastings et al Craniofacial features (Figure 1). Key diagnostic features are microcephaly and trigonocephaly. Trigonocephaly is usually due to prominence of the metopic ridge and bitemporal narrowing, with few having true metopic synostosis. It may not be apparent in the neonatal period and typically becomes less evident through early childhood. Microcephaly may be either primary or secondary, in either case tending to become increasingly pronounced through early childhood. Palatal abnormalities are a key feature. Sometimes cleft palate, or submucous cleft, is present but typically the palate is just abnormal with a high narrow grooved palate. Prominent eyes and hypoplastic supraorbital ridges, upslanting palpebral fissures, depressed nasal bridge and anteverted nares, facial flammeus nevus (which becomes less obvious with age), and low-set posteriorly-rotated ears are almost universal. A low anterior/temporal hairline is seen in most patients in the neonatal period but may become less pronounced with age. The older children show variability in their facial appearance. Some retain their appearance in infancy but with some features such as trigonocephaly, eye prominence and micrognathia becoming less prominent (eg patients 1, 2, 3, and 5). In others hypertrichosis and synophrys become more prominent and coarseness of the facial features can develop (eg patients 6, 7, and 14).

Hypertelorism, epicanthal folds, small mouth, broad alveolar ridges, and buccal frenulae (not seen in our series but reported previously) are found in some.

Ophthalmic features. Strabismus and retinal abnormalities are common (50%), as are anterior chamber abnormalities (20%), and high myopia (33%).

Growth and development. IUGR is seen in most (83%) and all who survive have feeding difficulties and fail to thrive. This is a key feature of BOS with gastrostomy feeding required from early infancy in all the patients we are aware of. In some this diminishes as a problem through childhood such that gastrostomy feeding may not be Hastings et al required in later childhood. Even in those that take food orally they are often reported as having relatively little interest in food and poor coordination of swallowing.

Despite this during childhood weight increases relative to height and is associated with central obesity in some, also demonstrated in the patient reported by Pierron et al [START_REF] Pierron | Evolution of a patient with Bohring-Opitz syndrome[END_REF] .

Neurological and musculoskeletal features. Structural brain abnormalities are commonly identified (70%) but non-specific. Reported findings include ventriculomegaly, delayed myelination, Dandy-Walker malformation, generalized atrophy, slender corpus callosum and brainstem, and neuronal migration abnormalities. One of the key features of BOS, which has been reported but not highlighted previously, is the unusual and characteristic limb posture these children adopt (Figure 2). This 'BOS posture' is recognized in all patients although is not entirely specific for BOS. It appears unrelated to overall tone, structural brain abnormalities, or joint contractures/dislocations. Older patients may have purposeful movement of the limbs but return to this posture as their resting state. Contractures are common (60%) and congenital dislocations are seen in many if investigated for (33%), with hip and radial head dislocations being the most common. In the older children joint problems remain problematic and may prove to be a significant cause of morbidity in these patients through adulthood. A few of our cohort have developed significant scoliosis although at present none of these have required surgery. Two patients have had femoral fragility fractures, in one patient attributed to renal tubular acidosis the other with osteopenia of unknown cause.

Internal organ abnormalities. Cardiac abnormalities are seen in almost half but are non-specific. Atrial septal defects, patent ductus arteriosus, and valvular abnormalities (most commonly pulmonary stenosis), have been reported. Septal hypertrophy was Hastings et al seen in two patients but, despite being severe enough to cause obstruction in the neonatal period, improved in both patients. Intra-abdominal organs are rarely noted to be abnormal. Bowel malrotation and inguinal hernias have been reported in some.

Morbidity and outcome.

There remains a high rate of infant mortality in this condition (40%), most commonly due to infections. If the early childhood period is survived then many of the common problems, such as feeding difficulties and recurrent infections, become less problematic. Severe to profound developmental delay is universal although there is some variability in terms of the level of communication and mobility achieved. One of the patients has made significant developmental progress in later childhood despite limited progress before this. Central obesity, sleep apnea, and type 1 diabetes have been noted in adolescence but numbers are too small to generalize whether these are more common in BOS.

The cause and inheritance of BOS are unknown. Apart from our published report of siblings with BOS 4 , patients 1 and 2 in this report, all cases have been sporadic. One patient came from a consanguineous family lending support to the likelihood of recessive inheritance. However some of the families without recurrence are large, for example patient 6 has eleven unaffected siblings. Presuming a genetic etiology, it therefore remains unclear whether BOS is caused by dominant mutations, with a germline mosaicism risk, or is a recessive disorder. It is also possible that there may be separate disorders with closely overlapping phenotypes but differing etiologies.

Patients have been identified with phenotypes resembling BOS due to chromosome abnormalities, the most similar being a patient with trisomy 3pter [START_REF] Mcgaughran | Trisomy of 3pter in a patient with apparent C (trigoncephaly) syndrome[END_REF] . Mutations in the TACTILE gene have been implicated as a cause of Opitz C syndrome, which has Hastings et al overlapping features with BOS, after one patient was found to have a balanced translocation causing disruption of this gene 10 and a missense mutation was subsequently identified in another 11 . However the clinical phenotype of these two patients is not typical of BOS and mutations of this gene have been excluded in patients with BOS, including several of those described here. This report helps to delineate the specific clinical phenotype of BOS, and provide insight into the frequency and spectrum of clinical problems encountered and their outcome. Identifying the cause of BOS is essential to clarify whether this clinical phenotype is a single entity, and indeed whether more varied phenotypes are due to the same cause but without the characteristic features. With the advent of nextgeneration sequencing technologies it is anticipated that the basis of BOS, and other rare disorders of unknown etiology, will become apparent in the near future and lead to an improvement in our understanding of the molecular processes underpinning them. Hastings et al

Titles and legends to figures:

  Hastings et al fractures. Her growth has shown the same pattern as her brother and at twelve years of age her height is 117 cm (<0.4 th centile), weight 25.2 kg (0.4 th -2 nd centile), and OFC 49.5 cm (<<0.4 th ). At twelve years of age she can sit unsupported at times and babbles but has no meaningful communication. Cranial MRI has shown a relatively small brain with expansion of the frontal horns of the lateral ventricles and nodular heterotopic grey matter in the wall of the left lateral ventricle. Echocardiogram showed a mild pulmonary artery stenosis and patent foramen ovale.Patient 3 was born at forty-one weeks gestation to non-consanguineous Caucasian British parents with a birth weight of 3.0kg (10 th centile). She had trigonocephaly, glabella nevus flammeus along with multiple capillary hemangiomas on the trunk, prominent eyes with hypoplastic supraorbital ridges, low-set ears with overfolded helices, broad alveolar ridges with a narrow V-shaped palate and abnormal uvula, deep transverse palmar creases, and long overlapping fingers and toes with partial 2/3 syndactyly. In recent years the eyes and trigonocephaly have become less prominent and the flammeus nevus has faded. She has developed small, puffy, cold feet, fetal finger pads have been noted, and she has slowly progressive scoliosis. She was hypotonic and had BOS posture along with congenital hip dislocation, radial head dislocation, and mild pectus excavatum. In infancy she had absence seizures and apneic episodes. Problems with recurrent chest infections in infancy and early childhood have since improved. Other problems included constipation, absent lacrimation, hyperhidrosis, and persistent primary dentition. She has unilateral squint, ptosis, and high myopia. Poor feeding requiring gastrostomy feeding and gastroesophageal reflux were problematic in early childhood but have since almost resolved. Indeed she is now overweight, at the age of fourteen years she weighs 54kg (50 th Hastings et al centile), having been below the 0.4 th centile, with height 141cm (<0.4 th centile) and OFC 50.5cm (<<0.4 th centile). Development was severely delayed but has made rapid progress in the past few years. She has been able to walk a few steps since the age of seven and despite no speech at ten years of age now has a good vocabulary and can form simple sentences. Recent problems with snoring and sleep apnea have been improved with adenotonsillectomy. At thirteen years of age she was diagnosed as an insulin-dependent diabetic (although she has a strong family history of this). Patient 4 was born at term to non-consanguineous Afro-Caribbean parents. Birth weight was 2.1kg (<0.4 th centile) with a length of 47cm (2 nd -9 th centile) and OFC of 34cm (25 th -50 th centile). She had prominent eyes with hypoplastic supraorbital ridges, hypertelorism, upslanting palpebral fissures, epicanthal folds, glabella nevus flammeus, depressed nasal bridge with anteverted nares, low-set ears, retrognathia, low temporal hairline, synophrys, hypertrichosis and deep palmar creases. BOS posture was present. Feeding was a significant problem and required gastrostomy feeding. She had high myopia. Secondary microcephaly developed, and at the age of two years and ten months her height was 73.7cm (0.4 th centile) and weight 8.7kg (<0.4 th centile). Echocardiogram demonstrated a patent ductus arteriosus and cranial MRI demonstrated a Dandy-Walker malformation. She died at four years and seven months old due to pneumonia. Patient 5 was born at forty-one weeks gestation by cesarean section for transverse lie to non-consanguineous Orthodox (Ashkenazi) Jewish British parents. She is the eleventh of twelve children with no similar problems noted in any other family members. Birth parameters were atypical with weight 3.83kg (75 th centile) and OFC Hastings et al 35.8cm (75 th centile). She had prominent eyes with hypoplastic supraorbital ridges, glabella nevus flammeus, depressed nasal bridge and anteverted nares, low-set posteriorly-rotated ears, small mouth, broad alveolar ridges with a high U-shaped narrow palate, submucous cleft, and choanal atresia, low temporal hairline, retrognathia, BOS posture, and bilateral knee dislocations and unilateral hip dislocation. She is gastrostomy fed due to feeding difficulties. Duodenal atresia was operated on at day three of life. At the age of five she takes small amounts of food orally but remains predominantly gastrostomy fed. Ophthalmic problems include high myopia and persistent pupillary membrane. At the age of five years her height is 99cm (0.4 th centile), weight 16kg (2 nd centile), having dropped below the 0.4 th centile in infancy, and OFC 47cm (<0.4 th centile). Development has been severely delayed, she has been able to stand from the age of three and at five is starting to communicate through picture signs and pointing. Echocardiogram showed mild pulmonary stenosis, ASD, and septal hypertrophy causing dynamic left ventricular outflow obstruction which resolved by six months of age.Patient 6 was born at term to non-consanguineous Caucasian British parents weighing 2.3kg (0.4 th centile). She had trigonocephaly, prominent eyes with hypoplastic supraorbital ridges, glabella nevus flammeus, broad alveolar ridges, low set dysplastic ears, retrognathia, low temporal hairline and hypertrichosis. In infancy she was hypotonic and displayed the BOS posture, along with having flexion contractures at wrists and elbows, bilateral radial head dislocation and bilateral congenital hip dislocation, overlapping fingers, partial 2/3 syndactyly and a mild pectus excavatum. She has had severe feeding problems along with failure to thrive requiring gastrostomy feeding, but no evidence of significant reflux. In infancy she had Hastings et al 'choking' episodes of uncertain cause and recurrent chest infections which improved by early childhood. Development is severely delayed but from eighteen months of age she has developed social behaviors. She has significant myopia and reduced hearing on the right side. At two and a half years of age her height is 73cm (<<0.4 th centile), weight 6.65kg (<<0.4 th centile) and OFC 43.6cm (<<0.4 th centile). Imaging has shown dextrocardia and cerebellar vermis hypoplasia, Dandy-Walker malformation, and thin corpus callosum.Patient 7 has previously been reported as part of a series discussing determination of pathogenicity in inherited cytogenetic abnormalities[START_REF] Bisgaard | Transmitted cytogenetic abnormalities in patients with mental retardation: Pathogenic or normal variants[END_REF] . She was born at thirty-five weeks gestation to non-consanguineous Caucasian Danish parents with a birth weight of 1.82kg (10th) and OFC 28.3cm (0.4 th centile). She had trigonocephaly with a fused metopic suture (requiring surgery at 9 months of age), prominent eyes, upslanting palpebral fissures, epicanthus, glabella nevus flammeus, high and narrow palate, hypertrichosis, low temporal hairline, synophrys, retrognathia, deep palmar creases, and sacral dimple. BOS posture and hypotonia were noted, with contractures at hips, knees, and ankles and development of a scoliosis. She had feeding difficulties and failure to thrive requiring gastrostomy feeding. Development was severely delayed with no development of speech or ability to sit. Recurrent chest infections and difficult to manage generalized seizures became apparent in infancy. Ophthalmic problems included a squint and poor vision with myopia. At the age of seven years her height is 98cm (<0.4 th centile), weight 15.3kg (0.4 th centile), and OFC 44.5cm (<<0.4 th centile). Cranial MRI showed generalized atrophy. Hastings et al Patient 8 was born at term to non-consanguineous Caucasian French parents. At birth she weighed 2.2kg (0.4 th centile), length was 43cm (<0.4 th centile), and OFC 31cm (0.4 th centile). She had trigonocephaly, glabella nevus flammeus, prominent eyes with hypoplastic supraorbital ridges, hypertelorism, epicanthal folds, broad alveolar ridges, low temporal hairline, hypertrichosis, and retrognathia. BOS posture was present along with long overlapping fingers. She had feeding difficulties and failure to thrive requiring gastrostomy feeding. Ophthalmic problems included squint and ptosis. At ten months height was 70cm (25 th centile), weight 8kg (9 th -25 th centile), and OFC 42cm (<0.4 th centile). Echocardiogram demonstrated pulmonary valvular stenosis and cranial MRI showed enlarged lateral ventricles. Bone age was advanced. She died at 11 months of age.Patient 9 was born at thirty-seven weeks gestation by cesarean section due to reduced fetal movements to non-consanguineous parents from Morocco. Birth weight was 2.4kg (9 th centile), length 46cm (9 th centile) and OFC 32cm (9 th -25 th centile).

Figure 1 :Figure 2 :

 12 Figure 1: Facial appearance of features of patients with BOS

ACKNOWLEDGEMENTS:

The authors would like to thank all of the patients and their families and Dr Kaname for analysis of the TACTILE gene in several patients.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.


Clément Quinton
email: clement.quinton@inria.fr

Sébastien Mosser
email: sebastien.mosser@inria.fr

Carlos Parra
email: carlos.parra@inria.fr

Laurence Duchien
email: laurence.duchien@inria.fr

Using Multiple Feature Models to Design Applications for Mobile Phones

Keywords: D.2.10 [Software Engineering]: Design-Methodologies, Representation; D.2.2 [Software Engineering]: Design Tools and Techniques-Computer-aided software engineering Software Product Line, Meta-Model, Feature Model, Application for Mobile Phones Smartphones, Meta-Model

The design of a mobile phone application is a tedious task according to its intrinsic variability. Software designers must take into account in their development process the versatility of available platforms (e.g., Android, iPhone). In addition to this, the variety of existing devices and their divergences (e.g., frontal camera, GPS) introduce another layer of complexity in the development process. These two dimensions can be formalized as Software Product Lines (SPL), independently defined. In this paper, we use a dedicated metamodel to bridge the gap between an application SPL and a mobile device one. This meta-model is also the support for the product derivation process. The approach is implemented in a framework named ApplIDE, and is used to successfully derive customer relationship management software on different devices.

INTRODUCTION

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Mobile phones, and smartphones in particular, are highly versatile ubiquitous systems. To automate the software development process for this particular subset of devices, it is important to take into account the different variations at the level of software, in the configuration of a mobile application product, but also at the level of hardware, and in particular, at the features supported by different devices running on different operating systems. For example, a functionality for a given application can be implemented in several ways, and, there can also be different mobile devices available (e.g., with or without frontal camera, GPS sensor). This combination of possibilities represents a major challenge for developers that want to reach a maximum of end-users.

One way to automate this development process is to use a Software Product Line (spl) approach. In an spl, multiple products are derived by combining a set of different core assets. One of the most important challenges of spl engineering concerns variability management, i.e., how to describe, manage and implement the commonalities and variabilities existing among the members of the same family of software products. A well-known approach to variability modeling is by means of Feature Diagrams (fd) introduced as part of Feature Oriented Domain Analysis (FODA) [START_REF] Kang | Feature-Oriented Domain Analysis (FODA) -Feasibility Study[END_REF] back in 1990. A fd typically consists of (i) a hierarchy of features, which may be mandatory (commonality) or optional (variability), and (ii) a set of constraints expressing inter-feature dependencies. Every feature can be realized by one or more assets. To build a product, the assets of different features are combined to obtain a software product. In this context, features can be seen as a way of modularizing a software product. Modularization, as introduced by Parnas [START_REF] Parnas | On the criteria to be used in decomposing systems into modules[END_REF], refers to the separation and localization of concerns. Separating such concerns helps specialists to focus on small modules of their expertise.

Contrarly to frameworks (see Sec. 5) that rely on codedriven approaches, we propose to use models as a support for (i) product derivation and (ii) consistency reasoning. Our approach use Model Driven Engineering (mde) and spl to bridge the gap between business variability and device variability. We propose to separate each domain with two different fds. For the business domain, we define an application dependent fd that represents the different configurations available for a given business. For the device do-main, we define an application independent fd that aggregates the variability of different and heterogeneous mobile devices. Using this separation we propose a derivation process that automates the development process through model merging and code generation, and that additionally analyses automatically the constraints of each product derived, with regard to the available mobile devices.

As a result, our derivation process allows developers to identify conflicts between a given product and the underlying hardware on which products are executed. To validate our approach, we use as a running example a Customer Relationship Management (CRM) product family, that contains up to 360 different product configurations. Nonetheless, the approach proposed here is not specific to CRM applications and can cover different domains.

The remainder of this paper is organized as follows. In Sec. 2 we present the MobiCRM example based on CRM applications. In Sec. 3 we describe the approach and the way mde and spl can be combined to support mobile application derivation. Sec. 4 presents the implementation and the results obtained from applying the approach. Finally, Sec. 5 compares our approach with close-related approaches in the literature and Sec. 6 concludes the paper.

MOTIVATION & CHALLENGES

In this section, we present a motivating example based on a CRM system. We discuss its representation as an fd and we describe the challenges our proposition faces.

Adaptation to heterogenous systems

In this paper, we propose a framework to manage the variability of mobile applications and devices to make them available to the largest number possible. To maximize the number of reachable end-users, a smartphone application (i.e., a product) has to be available on a maximum of mobile platforms. Unfortunately, a company has to develop concurrently several versions of the same piece of software, or complete software, targeting the different operating systems or devices. A piece of software, or a part of application specifications, can also be reused in another application, allowing time-saving and increasing development productivity. An alternative way is to develop it on a restricted set of platforms, accepting to lose end-users. This is not acceptable for a company that wants its application (e.g., products catalog, shops location) to be available for the largest set of end-users.

Running example

To illustrate our proposition, we introduce MobiCRM, a CRM application family for mobile devices, e.g., smartphones. A CRM system allows the user to have information about the company customers, (e.g., name, address). The main advantage of a CRM application dedicated to smartphones is that the information can be retrieved anywhere. For end-users, the entry point of the application is a login screen. There are different ways to access the user account. In other screens, the user retrieves a local or remote customers list depending on his/her login. He/she also interacts with these customers (e.g., calls them, sends them emails or SMS, takes a picture). As a result, the MobiCRM application family presents multiple variation points enabling a wide range for customization in order to address different user needs. We formalize these variations through the definition of a MobiCRM feature model, F M MobiCRM (Fig. 1).

The second variability dimension in mobile applications development is in the mobile devices and systems. For example, a MobiCRM product P1 = {Login, Remote, V ideo} will run on iPhone 4 but won't be able to run on older devices, e.g., iPhone 3GS. This kind of device does not have a front-facing camera to make a video call. Furthermore, even if the camera is available, devices running the Android operating system cannot provide this functionality on versions 2.3.3 and below. Being able to identify such inconsistencies gives strong support to designers as they are able to accurately know which device is able to run their software. We formalize these variations through the definition of a mobile device feature model, F M device (Fig. 2).

Challenges

We propose in this contribution to combine the F M device with a F M application , whatever the application (in our case MobiCRM), into a single solution for product derivation that gives strong support to mobile application developers. For this, we have to face several challenges: C1: Supporting application variability. The essential role played by the variability is to use it as a starting point for deriving products. The first challenge that has to be faced regards the definition of a process that uses as input any configuration obtained from the fd F M application and that results in a fully functional product.

C2: Supporting device variability. To maximize the number of reachable end-users for the different software products, it is necessary to identify the inconsistencies between such products and the mobile devices on which they have to be executed. The second challenge refers to the automatic detection of these inconsistencies, using as input the the products of the F M application , and a set of mobile devices like the ones represented by the F M device .

We propose the following framework to accurately address these challenges.

PROPOSAL

To face these challenges, we define a model-driven framework used to (i) support the derivation process and (ii) bridge the gap between an application fd and the mobile device one.

Merge iPhone

Android Windows Phone ...

Application Conguration Product Derivation

Analysis

Smartphone library (products)

Figure 3: Approach overview
We propose in a first step to tackle challenge C1 by (i) associating each application-dependent feature to a fragment of model, (ii) merge these fragments into a model that represents the expected product. In a second step, we handle challenge C2 by checking if this model conforms to a metamodel representing a given device. At the end, the application is generated using classic model transformations.

AppliDE, a Model-Driven Framework

We define AppliDE as a model-driven bridge to support the previously described approach. It models the intrinsic variability associated to mobile phones applications. Ap-pliDE is built upon industrial foundations, through an industrial transfer grant between INRIA Lille-Nord Europe and the UbInnov startup company. The main idea of this partnership is to reduce the time-to-market between the design of an application and its availability on multiple mobile devices.

AppliDE defines a meta-model to represent mobile applications. This meta-model allows one to model such a system in a platform independent way, which reifies smartphones application concepts. A model that conforms to this metamodel describes: (i) application functionalities (e.g., sending an email, retrieving device's location), (ii) data sources used to feed the application (e.g., local file, Web service invocation), and finally (iii) user-interface elements (e.g., buttons, inputs). A coarse-grained description of the AppliDE meta-model, called AppliDE MM, is depicted in Fig. 4 using the class-diagram formalism. In AppliDE, an application is the root element of the meta-model and is defined as a set of datasources and views. DataSources can be local (e.g., File) or remote (e.g., WebService). Views define several graphical objects (i.e., GUIObject) that can be both view or widget (e.g., Button, InputText). The Instruction concept represents a functionality (e.g., SendSMS, TakePicture, Call) that is triggered when an event occurs on a graphical element.

Supporting Product Derivation

To tackle the first challenge, we propose to bind each application-dependent feature to a fragment of an AppliDE model. Thus, a feature defined in the MobiCRM feature diagram holds as assets its corresponding model elements, that conforms to the AppliDE meta-model.

Modeling Assets.

Feature assets are defined in terms of model elements, with regard to the previously defined meta-model. Each feature is bound to fragments of model conform to the AppliDE meta-model. Such fragments can contain both structural (e.g., a button and its associated event) and behavioral (e.g., an action triggered by an event) elements.

The main asset is associated to the MobiCRM feature and with the mandatory features, in our case CustomerDB. It models the core of the application. It is composed by three Views, implementing the common user-interface provided to the user. The Authentication feature is associated to the Login-View that hosts the Login component, used to implement the authentication interaction. The CustomerDB feature is associated to a DataSource, which contains the customers. It also contains graphical elements used to display these customers and interact with them. Interact features (e.g., SendSMS) are associated to logical elements that enrich the available interactions with the customers.

We consider here two examples of model fragments extracted from the MobiCRM running example: (i) supporting customer interaction through SMS and (ii) user authentication. The former is implemented through a button and its associated event. A click on the button triggers a SMS to be send (Fig. 5a). The latter (Login feature) is associated to a fragment of model (depicted in Fig. 5b

Product derivation.

The derivation of a given product is thus supported by usual model composition techniques. We propose to use classical merge algorithms (e.g., Kompose [START_REF] Fleurey | A Generic Approach For Automatic Model Composition[END_REF]), denoted as µ in this paper. The algorithm takes as input the set of models to be merged, and produces as output the "merged" model, i.e., the expected product. In this example, we rely on a name-based matching mechanism, that is, the merge algorithm identifies elements with the same name and triggers their merge. We use a binding function β to associate a given feature F and its associated model elements {me1, . . .}. Considering a product as a set of selected features p = {F1, . . . , Fn}, we obtain its associated model mp Let us now consider a product that includes customer interaction with SMS, and authentication (Fig. 7). Thus, the product is composed by SMS and Login features, in addition to the application core: p1 = {Login, SM S, M obiCRM }. The composed model (i.e., the derived product) is depicted in Fig. 8.

Application generation.

AppliDE allows the user to generate applications source code for several mobile devices e.g., smartphones. We rely on model-transformation techniques to support this generative process. The AppliDE model associated to a given product (i.e., obtained from the derivation process) is transformed into its representation with regard to a given execution platform. Thus, a given model m can be transformed into an iPhone application ai thanks to a dedicated modeltransformation τi: ai = τi(m). To support a new execution platform pl, one defines a new transformation τ pl , and adds

Bridging the gap

The previous subsection illustrates how features defined in the application fd are associated to fragments of models. To tackle challenge C2, we need to bridge the gap between this fd and the mobile device one. Following an endogenous approach, we rely on the AppliDE meta-model to implement such a bridge: application-dependent features holds fragments of model as assets, and device-dependent features are associated to meta-model concepts. provided by smartphone companies. The application designer uses the AppliDE MM to configure an application, whatever the application (in our case MobiCRM), and define its associated features. Considering AppliDE as the bridge that connects all the different artifacts, an application designer benefits from the two previously described advantages: (i) automatic generation of the executable applications and (ii) device analysis.

The AppliDE MM can be pruned [START_REF] Sen | Meta-model pruning[END_REF] into a new metamodel called Functionality MM. For a given device d, a transformation extracts all the AppliDE MM concepts associated to the Functionality features of the device fd to obtain the Functionality MM. If a given model m derived from a product p conforms to the Functionality MM, then one can say that p can be executed on d.

Sketching formal analysis.

Thus, we can reduce the analysis of an application product to a type-conformance problem. On the one hand, let p ∈ F M MobiCRM a valid product, and mp its associated merged model. It requires a set of functionalities (denoted as Fp), e.g., SMS sending, GPS sensor. These functionalities are implemented in AppliDE through the concepts defined in the meta-model (e.g., the SendSMS instruction). Thus, one can understand Fp as the set of concepts used by the model elements contained by mp. On the other hand, let us consider now d a given device, i.e., a product defined according to F M device . According to the previously given definition, the assets associated to each feature are concepts defined in AppliDE. Thus, the assets A d associated to d represent a set of concepts that one can use in an application to safely execute it on d (a pruned meta-model [START_REF] Sen | Meta-model pruning[END_REF]). An immediate analysis to assess if a given product can be executed in a given device is to check if Fp ⊆ A d . Nevertheless, as we reduce the analysis as a type conformance problem, elaborated techniques from this dedicated research field can be used to tackle this goal.

For instance, consider the following configurations:

p1 = {SMS, Email} p2 = {SMS, V ideo} d1 = {iP hone 3, SMS, Email, Call Classic} d2 = {iP hone 4, SMS, Email, Call V ideo}.
Using d1 in our transformation, the Functionality MM (called MMFunc1) obtained after pruning holds SMS, Email and Call Classic as concepts. Using d2, it (MMFunc2) holds SMS, Email and Call Video concepts. Let m1 the model derived from p1 and m2 the model derived from p2. m1 conforms to MMFunc1 and MMFunc2 while m1 conforms only to MMFunc2 We can deduce that the application associated to the p1 configuration can be executed on d1 and d2 while the one associated to the p2 configuration can only be executed on d2.

This analysis allows us to determine on which device(s) a MobiCRM product is able to run. Assuming the availability of a device catalog (expressed using F M device ), the result of such analysis gives to the designers the set of device products a given MobiCRM application is consistent with and consequently a set of "real" devices that can execute the application. Thus, one can immediately know the market share reachable by his/her current product.

VALIDATION

In this section we present the results obtained from the experimentation, as well as the implementation details of AppliDE.

Experimentation

As stated before, the MobiCRM fd represents 360 different product configurations. To validate our approach, we have used these configurations as input in our derivation process. In each case, we successfully obtain a complete product, and additionally, depending on the case, we verify if the product can be executed on a given device.

Consider the products we have presented in Sec. 3.3. For the product p1, after the merging and the generation of code, we obtain a fully functional software product of around 1500 lines of code. The analysis shows that this product is compatible with the devices d1 and d2. For the product p2 we also obtain a fully functional software product with a number of lines of code similar to p1. However, in this case, we find an inconsistency with the specified device. The video calling feature can only be used on devices with a frontfacing camera. The functional meta-model obtained after the pruning process does not contain the meta-classes for video calling. This product however, can be executed in devices that support such functionality like the iPhone 4 and several Android smartphones. In total, 120 out of the 360 configurations cannot be executed in the iPhone 3 (device d1).

Implementation

Regarding the implementation, the AppliDE framework relies on Eclipse Modeling Framework (EMF) [START_REF] Steinberg | EMF: Eclipse Modeling Framework (2nd Edition)[END_REF] metamodels, which is one of the most widely accepted metamodeling technologies. The EMF provides code generation facilities for building tools and other applications based on a structured data model. The XMI format is used to support model persistence.

The feature diagrams for both MobiCRM and the mobile devices have been described using the FAMILIAR environment [START_REF] Acher | A domain-specific language for managing feature models[END_REF] (Fig. 10). We also used the FAMILIAR tools to compute the number of available configurations.

RELATED WORKS

Multi-view modeling approaches are used to support the design of complex systems, based on different points of views. In Ram (Reusable Aspect Models [START_REF] Klein | Reusable Aspect Models[END_REF]), one can use both class diagrams and sequence diagrams to design a system. The approach supports the concurrent weaving of these two kinds of artifacts, and maintains consistency. The class diagrams are merged according to [START_REF] Reddy | Directives for Composing Aspect-Oriented Design Class Models[END_REF], and sequence diagrams according to Klein's method [START_REF] Kienzle | Aspect-Oriented Multi-View Modeling[END_REF]. The Theme/Uml approach [START_REF] Carton | Model-Driven Theme/UML[END_REF][START_REF] Gomaa | A multiple-view meta-modeling approach for variability management in software product lines[END_REF] are another interesting multi-view modeling approach [START_REF] Barais | Composing Multi-View Aspect Models[END_REF], very similar to Ram. Our proposition is clearly complementary with these ones, as we bring to the modelcomposition universe the architectural knowledge that a feature diagram reifies. Moreover, these powerful mechanisms can be reused in our approach, as we are based on the same approach of name-matching mechanism for the underlying composition.

Code-driven approaches, such as Rhomobile, PhoneGap, Titanium Mobile or MoSync translate HTML and JavaScript code into iPhone or Android source code. Contrarily to these syntactically-driven approaches, we propose to use models as a reasoning support. Our meta-model bridge the gap between two software product lines, thus ensuring their consistency and co-evolution. Dhungana et al. [START_REF] Dhungana | Structuring the modeling space and supporting evolution in software product line engineering[END_REF] support evolution of complex systems by defining model fragments representing subsystem parts and checking their consistency. The complete model is then obtained by merging fragments. In our approach, we systematically bind a model fragment to a given feature, introducing feature expressiveness into these artifacts. The originality is to ensure product (merged model) consistency according to two dimensions: application and mobile device variability.

With regard to derivation safety, the Mata approach [18] supports the weaving of models aspects using a graph-based approach. This approach supports powerful conflict detection mechanisms, used to support the "safe" composition of models [START_REF] Mussbacher | Semantic-Based Interaction Detection in Aspect-Oriented Scenarios[END_REF]. The underlying formal model associated to this detection is based on critical pair analysis [START_REF] Heckel | Confluence of Typed Attributed Graph Transformation Systems[END_REF]. Initially defined for term rewriting system and then generalized to graph rewriting systems, critical pairs formalize the idea of a minimal example of a potentially conflicting situation. This notion supports the development of rule-based system, identifying conflicting situations such as "the rule r will delete an element matched by the rule r " or "the rule r generates a structure which is prohibited according to the existing preconditions". These mechanisms were demonstrated as relevant to identify composition conflicts in the software product line domain [START_REF] Jayaraman | Model Composition in Product Lines and Feature Interaction Detection Using Critical Pair Analysis[END_REF]. Unlike these approaches, in our case we do not focus on the derivation safety of a given product configuration. We rather aim at identifying the compatibility between a given software product and a set of available mobile devices. This is done by specifying separately the application and device variability, and pruning the application meta-model with regard to each specific device configuration.

CONCLUSION & FUTURE WORKS

In this paper, we have presented a model-driven approach that supports mobile application development, ensuring application products to be platform independent. We start with a product family represented through a fd. For every feature in the fd, we define an associated model fragment defining an architectural element. We use the AppliDE framework and in particular, the meta-model to bridge the gap between the software spl and the device one. The functionality features of the device fd are associated to metamodel concepts. If the merge of fragments model conforms to the meta-model we can deduce which device is able to run the application.

Our approach faces the challenges identified in Section 2. For the first challenge regarding the variability of the application, we have presented the merging of fragments as well as the generation of code for different mobile platforms. For the second challenge, regarding the device variability, we have defined a pruning process that creates a reduced version of our application meta-model. This meta-model is used to check if the product being derived can be executed in a given hardware. To validate the approach, we have used it to develop the MobiCRM product family. The results of our experimentation show that our approach helps to deal with the variability of a MobiCRM product (360 different configurations) and the intrinsic variability of mobile phone devices. Furthermore, using the model-driven process provided by the AppliDE framework, we are able to generate fully functional software products on two different mobile platforms.

For future work, we think that our approach can be extended to face the challenge of inconsistency resolving, helping the developer to reach the maximum of end-users. Some decision rules could be added to the framework and proposed to the developer, such as a feature that is only available on a few systems in order to reach a bigger number of end-users (e.g., if one removes the Video feature from an iPhone 4 application, one will reach all the iPhone 3 owners).

Figure 1 :

 1 Figure 1: F M MobiCRM

Figure 2 :

 2 Figure 2: F M device (extract)

Figure 4 :

 4 Figure 4: The AppliDE Meta-Model (extract)

Figure 5 :

 5 Figure 5: Features and associated model elements

Figure 6 :

 6 Figure 6: The MobiCRM application (extract)

Figure 7 :Figure 8 :

 78 Figure 7: Merging assets (model fragments)

Figure 9 :

 9 Figure 9: Consistency verification process overview

FM

Figure 10 :

 10 Figure 10: MobiCRM fd defined with FAMILIAR

Figure 11 :

 11 Figure 11: The Android template associated to the SMS feature This choice brings high flexibility to the framework. Indeed, one can target a new platform by adding corresponding source code generator templates (e.g., for Windows Phone).

We rely on usual techniques (e.g., model-to-text template engines) to implement the model-transformation chain, which eases the development of new ones and their adoption by engineers in an industrial context.

Acknowledgments

This work is supported by Ministry of Higher Education and Research, Nord-Pas de Calais Regional Council and FEDER through the Contrat de Projets Etat Region Campus Intelligence Ambiante (CPER CIA) 2007-2013.


L Biadala

Ph. Tamarat Y Louyer

B Lounis
email: b.lounis@cpmoh.u-bordeaux1.fr

Direct observation of the two Lowest Exciton Zero-Phonon Lines in Single CdSe/ZnS Nanocrystals

We report a spectroscopic study of highly photostable individual CdSe/ZnS colloidal nanocrystals. At low temperature, photoluminescence spectra display two sharp zero-phonon lines which we attribute to the radiative recombination from the two lowest levels of the bandedge exciton fine structure. For the first time, resonant photoluminescence excitation spectra of these lines is performed, and spectral diffusion broadening of 10 µeV is measured over integration times of 100 ms, corresponding to an optical coherence lifetime longer than 100 ps.

Over the past decade, interest in low-dimensional, mesoscopic systems, such as semiconductor nanocrystals (NCs), has grown dramatically [START_REF] Efros | Semiconductor Nanocrystals: From Basic Principles to Applications[END_REF]. These NCs bridge the gap between single molecules and the solid state, thereby offering the opportunity to study the evolution of bulk properties. Additionally, their size-dependent optical properties due to quantum confinement make them ideal candidates for tunable absorbers and emitters in applications such as nanoscale electronics [START_REF] Colvin | [END_REF][3][4], laser technology [5] and biological fluorescent labelling [6,7]. Chemically synthesized CdSe NCs are interesting for many of these applications due to their bright luminescence, which is size tunable across the visible spectrum [8].

A detailed understanding of the band-edge exciton fine structure is crucial for these applications. It is well established, on both theoretical and experimental grounds [START_REF] Klimov | Semiconductor and Metal Nanocrystals:Synthesis and Electronic and Optical Properties[END_REF], that in spherical or nearly spherical colloidal CdSe NCs the band-edge exciton (1S 3/2 -1S e ) is split by the intrinsic electron-hole exchange interaction and by the crystal field of the hexagonal lattice into five energy levels [START_REF][END_REF]11]. The exciton ground state F , which is often referred to as the dark exciton, has a total spin projection on the crystal hexagonal axis ) exciton state A .

Studies of the photoluminescence (PL) spectra and decay as a function of temperature and external magnetic fields [START_REF][END_REF][12][13][14][15] showed that the luminescence arises from the two thermally mixed lowest states of the band-edge exciton fine structure. At liquid helium temperatures, the luminescence of NCs with strong confinement (radius smaller than 3 nm) originates primarily from the long-lived (hundreds of nanoseconds) dark exciton state, because the bright-to-dark spin-flip rate is much larger than the radiative recombination rate of the bright exciton state [START_REF][END_REF]14,16]. The PL spectra display a single zero-phonon line (ZPL) [17][18][19] which is therefore attributed to the weakly allowed radiative recombination of the dark state.

In this work, we report a cw and time resolved spectroscopic study of individual CdSe NCs with a weaker exciton confinement (radius of ~4 nm). At low temperature, these NCs display emission spectra with two sharp ZPLs that we attribute to the radiative recombinations from the two lowest exciton levels (bright and dark), as a consequence of a weak spin-flip rate between these two states. We also performed, for the first time, resonant photoluminescence excitation of these transitions and obtained excitation lines as narrow as 10 µeV over an integration time of 100 ms. NA objective which is inserted in a helium cryostat together with the sample and a piezo-scanner. The emitted photons are filtered from the scattered excitation light by a bandpass filter ( 60 nm FWHM) and sent to a single-photon-counting avalanche photodiode and a spectrometer (1800 lines/mm grating) equipped with a liquid-nitrogen-cooled camera. The PL decay measurements are performed with a conventional time correlated single photon counting setup, using a pulsed laser source (optical parametric oscillator at 570 nm, 250 fs pulse width, 2.5 MHz repetition rate). The instrumental response function of the system was 300 ps FWHM. The high stability of our experimental microscopy setup allows the study of the same single NC over a wide range of temperatures and over several days. At 2K the luminescence intensity of most of the single NCs is remarkably stable and did not show any blinking, as exemplified by the time trace of a single NC presented in the inset of Fig. 1(a). Shot-noise-limited intensity fluctuations were measured over minutes of acquisition times. Figure 1(a) shows the PL spectrum of this NC recorded over one minute at a weak cw excitation intensity of 60 W/cm 2 . Interestingly, the spectrum displays two peaks 0.7 meV apart around 2.027 eV with resolution-limited linewidths (~300 µeV). The intensities of the peaks have a linear dependence on the excitation power, excluding any biexcitonic origin for this structure. A similar spectral structure was found for tens of single NCs. These peaks which are labelled A and F are attributed to the bright and the dark exciton radiative recombination ZPLs, respectively. Indeed, the peak separation is in agreement with the energy splitting of ~ 1 meV between A and F expected from the theoretical analysis of the exciton fine structure for 4 nm NCs [11]. The peak around 2.00 eV, which is 26.6 meV red-shifted from the main peak F, corresponds to the longitudinal optical (LO) phonon replica of the F state ZPL [17]. One can also distinguish a weaker structure denoted Ac, 0.8 meV red-shifted from F. We attribute this feature to a confined acoustic phonon sideband due to electron coupling to the acoustic radial breathing mode of the NC [19,20].

Samples of

In order to confirm the attribution of the emission peaks A and F, we studied their temperature evolution on the same NC, as shown in Fig. 1(b). Comparing spectra from 2 K to in agreement with the picture of a phonon mediated thermal mixing between the bright and the dark excitons. The broadening of the peaks is attributed to the population of acoustic phonon modes that rises with temperature, washing out the two ZPLs above 15 K.

The striking observation of the two ZPLs in the PL spectrum points to a reduced spinflip relaxation rate in these relatively large NCs, in contrast to small NCs where only a single ZPL (attributed to the F state, the thermal population of the A state being negligible) is observed. As a typical example, Fig. 1(c) shows the emission spectrum of a ~2.2 nm radius single NC at 2K. In this case, the integrated intensity of the ZPL is weaker than that of the acoustic phonon sideband, supporting the assumption that the radiative recombination from this state occurs preferentially with emission of phonons. Yet the origin of the dark exciton purely radiative recombination is still an open question. Semiempirical pseudopotential calculations [21] show that this recombination can be due to a surface-induced mixing of dark and bright exciton states. However, spectroscopic investigations led on CdSe NCs with different coating shells argue against the involvements of surface states, and suggest a mechanism intrinsic to the CdSe NCs [22]. Beyond the effective mass approximation, semiempirical tight-binding calculations that incorporate spin-orbit interactions [23] provide an explanation for the finite lifetime of the dark exciton and for its decrease with the NC radius [13,22]. Other recombination mechanisms may involve spin-flip transitions of the F state induced by paramagnetic defects in the lattice [11], or by the hyperfine interaction with unpolarized nuclear spins [24].

We also performed ultra-high resolution resonant photoluminescence excitation (R-PLE) of the two lowest energy ZPLs of a single NC at 2 K. PLE of upper levels of the bandedge exciton fine structure has already been measured on individual NCs and led to linewidths of a few meV [25]. Our new method consists in sweeping the frequency of a cw single mode tunable dye laser (spectral resolution ~ 4 neV) around the central frequencies of the A and F ZPLs, while collecting the red-shifted luminescence of the LO mode through a long pass filter on an avalanche photodiode. The relative weight of the LO-phonon line compared to the ZPL is of the order of 10%, giving a count rate of thousands per second sufficiently high to record R-PLE spectra. The R-PLE lines of a single NC are displayed in Fig. 2 (a-c) at a very low excitation intensity of about 1 W/cm 2 , (well below saturation, the signal being linear with intensity). In the case of single scan spectra [see Fig. 2(b,c)] recorded on a time scale of 100 ms, which corresponds to the passage time through resonance, the lines are extremely sharp and display linewidths of the order of 10µeV. Such linewidths rule out any correspondence with a phonon band for these lines [25], and therefore supports their attribution to purely electronic lines. These spectra display the narrowest homogeneous lines ever recorded on individual CdSe NCs, setting a lower bound of 100 ps for the optical coherence lifetime of the two lowest energy bright and dark excitons over a time scale as long as 100ms. Dephasing times of the same order of magnitude, but over a time scale of 200 µs, were recently reported by Coolen et al. using a newly developed photon-correlation Fourier spectroscopy technique [26]. Even if the NCs used in our studies seem to present an efficient surface passivation, R-PLE spectra recorded on several frequency sweeps display a broadening with the number of scans (~ 30µeV, after 10 scans) [Fig. 2 In order to explain our observation of the lowest energy ZPL doublet, we need to determine the relaxation rates involved in the emission dynamics. Therefore, we studied the temperature dependent PL decay of many single NCs. Figure 3(b) shows the PL decay of a single NC for various temperatures ranging from 2 K to 105 K. At temperatures below 7 K, the decay shows a biexponential behavior with a fast decay occurring within few ns and a much slower one with a lifetime 1   L in the 200 ns range. At higher temperatures (>10 K), 1   L shortens and the fast component vanishes completely, as previously reported [14]. We explain the PL behavior with a three-level model illustrated in Fig. 3(a). It takes into account the zeroexciton ground level, G , and the two lowest states of the band-edge exciton fine structure, A and F , with respective radiative rates A  and F  . Thermal mixing between these excitonic states occurs via emission and absorption of acoustic phonons from a mode whose energy E  matches the energy splitting between A and F states. The , where 0  is the zero-temperature relaxation rate of the

F A  transition and     1 exp 1    T k E N B B
is the Bose-Einstein phonon number at temperature T. In order to obtain a sufficient counting rate from a single NC, a relatively high laser repetition rate (2.5 MHz) is needed to record the PL decays. As a consequence, the excitation period is not much longer than the lifetime at low temperature. A contribution from a multiexcitonic recombination to the short time decay component cannot be completely excluded, even if the excitation intensity was kept low enough to ensure a mean number of exciton per pulse smaller than one. We therefore used the temperature dependence of the long time decay component and PL spectra to determine the relaxation rates. The decay rate of the long time component is deduced from the solutions of rate equations for the excitonic states populations: ). From the fit we obtain the bright state decay rate A  ~0.16 ns -1 , and 0  = 0.87 ns -1 . This weak spin-flip relaxation rate suggests the existence of a phonon bottleneck effect [27][28][29][30] for large NCs where the energy splitting E  is in the meV range, and explains the observation of the ZPLs of both A and F states in the PL spectrum of the NC. Furthermore, the relative weight of the emission rate from the state A can be estimated within the frame of our three-level model. Assuming that the two lowest excitonic levels have identical quantum yields and are equally populated from the high energy continuum states [14,31], this weight is

                                             T k E T k E B F A B F A L 2 sinh 2 cotanh 2 1 2 2 0 2 0 0  (1).
  0 2     A A /
in the low temperature regime. Using the relaxation rates deduced from the PL decay analysis, we obtain a weight of ~ F state. As we raise the temperature from 2 K to 10 K, the weight of the LO-phonon band significantly drops with respect to the high energy structure (which includes A and F ZPLs and Ac) [see inset of Fig. 4 (a)]. A similar behavior has been reported on ensemble measurements using fluorescence line narrowing spectroscopy [32]. Our thermal mixing model now explains this observation as follows: An increase of temperature will lead to an increase of the A state ZPL and therefore to a decrease of the LO-phonon band relative weight. Most of the studied NCs displayed a less efficient LO-phonon coupling for the bright exciton than the dark. Note that we found few NCs show similar LO-phonon couplings for both states, as exemplified in Fig. 4 (b). As expected from the model, the weight of the LOphonon replica does not depend on temperature [see inset of Fig. 4 (b)] in this case.

In summary, we observed in the low temperature emission spectra of single CdSe/ZnS NCs two ZPLs attributed to the radiative recombinations from the two lowest levels of the band-edge exciton fine structure. This observation points to a size dependence of the brightto-dark spin-flip rate. From the analysis of the temperature dependent PL decay rate and the PL spectrum with a three-level model, we can deduce the bright exciton radiative rate as well as the bright-to-dark spin-flip rate for a single NC. We also performed for the first time ultrahigh resolution R-PLE of the two lowest energy ZPLs for a single NC at 2K. This experiment sets a lower bound of 100 ps for the optical coherence lifetime of the two lowest energy bright and dark excitons over a time scale as long as 100 ms. Further investigations will aim at determining the dependence of the exciton relaxation rates on the NC size or applied magnetic fields.

  meV below the lowest-energy optically active (

  CdSe/ZnS core-shell NCs (Qdot655 streptavidin conjugates from Quantum Dot Corp., peak emission at 655 nm at room temperature) are prepared by spin coating clean glass coverslips with a nanomolar solution of NCs in polyvinyl alcohol. A homebuilt scanning confocal microscope is used to image single NCs excited with the 532 nm line of a cw frequency doubled Nd:YAG laser. It is based on a 95 . 0

  (a)] due to residual spectral diffusion, i.e. random spectral shifts of the transition frequencies due to fluctuations in the local nano-environment.

Figures 3 ( 1 

 31 Figures 3(b) and 3(c) show the temperature dependence of

  Fig. 1(a) or Fig. 4(a), do not display LO-phonon replica of the bright state ZPL at 2 K, whereas the weight of the LO-phonon band represents ~ 20 % of the total emission from the darkF state. As we raise the temperature from 2 K to 10 K, the weight of the LO-phonon band significantly drops with respect to the high energy structure (which includes A and F

FIG. 1 .

 1 FIG. 1. (a) PL spectrum at 2 K of a single NC (radius ~4nm), recorded with an integration time of 60 s and at an excitation intensity of 60 W/cm 2 . The peaks labelled F and A are attributed to the ZPLs of the dark and the bright excitons respectively (see the text). The peak LO-F is a LO-phonon replica of the peak F. The Ac structure is a confined acoustic phonon sideband of the peak F. The inset shows the time trace of the integrated PL intensity recorded simultaneously with the PL spectrum for this NC. The distribution of the photon counts per bin (100 ms) shows that the intensity fluctuations are shot-noise limited. (b) A Zoom of the PL spectrum shown in Fig 1(a), recorded at various temperatures ranging from 2K to 20K. (c) A typical PL spectrum at 2K for a ~2.2 nm radius individual NC. The presence of a single sharp line is attributed to the F state ZPL.

FIG. 2 .FIG. 3 .FIG. 4 .

 234 FIG. 2. R-PLE and PL spectra of a single NC at 2 K. (a) R-PLE spectrum of the F state ZPL, recorded on ten frequency scans at an excitation intensity of 1 W/cm 2 . The duration of one scan is 1 s. (b) R-PLE spectrum of the F state ZPL recorded on a single scan, showing a

We thank Alexander Efros for fruitful discussion and Jonah Shaver for careful reading of the manuscript. This research was funded by the Région Aquitaine, the Agence Nationale pour la Recherche (ANR) and the French Ministry for Education and Research (MESR).