Emilie Sundqvist

Maria Bäärnhielm

Lars Alfredsson

Jan Hillert

Tomas Olsson

Ingrid Kockum
email: ingrid.kockum@ki.se

Confirmation of association between Multiple Sclerosis and CYP27B1

Keywords: Multiple Sclerosis, CYP27B1, Association study, vitamin D, Case control study, meta analysis

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Introduction

Multiple Sclerosis, MS (OMIM #126200) is an inflammatory disease, characterised by demyelinating lesions in the brain or spinal cord, axonal loss and progressive build-up of sclerotic plaques. It has a complex disease etiology where both genetic and environmental factors influence disease susceptibility. To date the strongest genetic factor found is the HLA-DRB1*15 haplotype (DQB1*0602, DQA1*0102, DRB1*1501, DRB5*0101). Many other genes with more modest effects on disease risk have been found through candidate gene approaches and genome wide association studies.

There is an apparent geographical pattern in MS prevalence, with increasing prevalence with increasing latitude. This has been observed both in the US, Europe, and Australia 1 . The level of solar radiation and UV radiation has been implicated in causing this difference in prevalence [START_REF] Acheson | Some comments on the relationship of the distribution of multiple sclerosis to latitude, solar radiation, and other variables[END_REF][START_REF] Van Der Mei | Regional variation in multiple sclerosis prevalence in Australia and its association with ambient ultraviolet radiation[END_REF] . There is also a month-of birth effect in MS susceptibility, with fewer cases born during the autumn months and more cases born in the spring compared to healthy controls [START_REF] Willer | Timing of birth and risk of multiple sclerosis: population based study[END_REF][START_REF] Bayes | Timing of birth and risk of multiple sclerosis in the Scottish population[END_REF] which could be linked to differences in climate during the year, including sun exposure.

Recently the Australian and New Zealand Multiple Sclerosis Genetics Consortium, ANZgene, published their findings from a genome wide association study in a total of around 2900 cases and 5700 controls. Besides confirming the association of a number of previously known genes, they also found associated SNPs on chromosomes 12q13-14 and 20q13. The 12q13-14 region includes 17 known genes, including CYP27B1 (cytochrome P450 family 27 subfamily B peptide 1). In the combined cohort, rs703842, which lies 1.76 kb upstream of CYP27B1 was associated with MS (p = 5.4 x 10 -11 ) [START_REF] Australian | Genome-wide association study identifies new multiple sclerosis loci on chromosomes 12 and 20[END_REF] .

Association to CYP27B1 has been seen in autoimmune diseases; rs4646536 (+2838T>C) which is in high LD with rs703842, and rs10877012 (-1260C>A) have previously been associated to type 1 diabetes 7,8 , Hashimoto's thyroiditis, and Graves' disease [START_REF] Lopez | A promoter polymorphism of the CYP27B1 gene is associated with Addison's disease, Hashimoto's thyroiditis, Graves' disease and type 1 diabetes mellitus in Germans. (B)[END_REF] . Rs4646536 programming of antigen-specific response, so called T-cell homing [START_REF] Van Etten | Regulation of vitamin D homeostasis: implications for the immune system[END_REF] . Overall, the findings support a suppressive role of vitamin D3 on the adaptive immune system.

Sundqvist et al. Confirmation of association between Multiple Sclerosis and CYP27B1

Low levels of vitamin D in serum are associated with a higher risk for developing MS [START_REF] Munger | Serum 25-hydroxyvitamin D levels and risk of multiple sclerosis[END_REF] and several studies show that MS patients have lower levels of 25-OHD 3 compared to controls 11 . A recent study showed that high circulating 25-OHD 3 levels among MS patients was associated with improved regulatory T cell function. It also had an impact on the T helper cells, shifting the balance in favor of Th2 cells [START_REF] Smolders | Vitamin D status is positively correlated with regulatory T cell function in patients with multiple sclerosis[END_REF] .

Based on the collective previous data on vitamin D, sun exposure, and inflammatory diseases, we decided to study if variants in the CYP27B1 gene were associated to MS in a Swedish population. SNP markers were selected for genotyping by running the tagging algorithm [START_REF] De Bakker | Efficiency and power in genetic association studies[END_REF] implemented in Haploview 3.32 based on data from the HapMap project using r 2 =0.9 and LOD 3.0. (http://www.hapmap.org/, release 21/phaseII, NCBI B35 assembly, dbSNP b125).

Materials and methods

Dataset

Two additional SNPs were included because of either reported association to type 1 diabetes (rs10877012) or because of a proposed involvement in MS susceptibility (rs4646536) [START_REF] Lopez | CYP27B1 polymorphisms variants are associated with type 1 diabetes mellitus in Germans[END_REF][START_REF] Orton | Evidence for genetic regulation of vitamin D status in twins with multiple sclerosis[END_REF] . A complete list of included SNPs is shown in table 1.

Genotyping

Two methods were utilized for allelic discrimination in the Swedish cohorts (see table 1).

Rs10877012 and rs4646536 were genotyped using Taqman SNP ® assays from Applied Biosystems as described previously [START_REF] Ekelund | Elevated expression and genetic association links the SOCS3 gene to atopic dermatitis[END_REF] . 6 SNPs, including rs4646536 were genotyped in all cohorts using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Sequenom Inc., San Diego, CA, USA), as described previously [START_REF] Roos | The interleukin 23 receptor gene in multiple sclerosis: a case-control study[END_REF] . Primer sequences are available upon request. One of the tagSNPs (rs11172327), which only tagged itself, was excluded from the analysis because of low success rate <75%, however this marker was also monomorphic in our samples.

Imputation and statistical analysis

Association of genotypes in the Swedish samples was tested using the model command in PLINK v1.06 (http://pngu.mgh.harvard.edu/~purcell/plink/index.shtml). Imputation was performed using Mach 1.0 20 accessed through the HapMap Genome Browser (Phase 3genotypes, frequencies and LD). The CEU population was used as reference population; the region was set to chromosome 12, 56,430,000 kb to 56,470,000 kb. rs8176345 was excluded from the imputation because it was not in Hardy-Wienberg equilibrium among controls. All other settings were kept at default. A fixed effects (Mantel-Haenszel) metaanalysis was performed using the rmeta package and the meta.MH command in R 2.8.0, a joint p-value was obtained from Unphased using study population as a covariate [START_REF] Dudbridge | Likelihood-based association analysis for nuclear families and unrelated subjects with missing genotype data[END_REF] .

Results

Seven SNPs were genotyped in the Swedish cohorts, described in more detail in table 1 (also see supplementary table 1 for detailed information for each of the three cohorts). All SNPs, except rs8176345 were in Hardy-Weinberg equilibrium among the unaffected controls. Three SNPs genotyped in the Swedish cohorts were associated to MS, rs4646536, rs10877012 and rs10877015, with p-values of 0.01, 0.01 and 3.5 x 10 -3 , respectively. They all had a reduced minor allele frequency among cases, with odds ratios of 0.88, 0.88 and 0.87, respectively for the minor allele. When we looked at the linkage disequilibrium in the region (figure 1), and defined the blocks either by the four gamete rule or solid spine of linkage disequilibrium there were two associated haplotypes, a CTACGA-haplotype (p = 8.5

x 10 -3 ) and a CTGCTG-haplotype (p= 7.8 x 10 -3 ). Defining the blocks by the confidence interval method gave similar results.

Imputed SNP and combined analysis

The most associated SNP from the ANZgene study located in the 12q13-14 region was rs703842, at position 55,449,006, between rs10877012 and rs10877015, run in our own samples. Genotypes from 1759 controls and 2158 Swedish cases were imputed. Other SNPs found in the ANZgene paper could not be imputed in our material. Rs703842 was found to be significantly associated (table 1), with a p-value of 0.02, and an odds ratio of 0.89 (0.81 -0.98 95% CI) for the minor allele, showing the same protective effect found in the ANZgene datasets.

For the combined analysis, genotypes from a total of 6032 cases and 7482 controls were available after imputation (see table 2). The results from the meta-analysis can be seen in figure 2. The combined OR for rs703842 was 0.83 (0.79 -0.88 95% CI). The overall association was significant, p = 5.1 x 10 -11 .

Discussion

In this study we tested the association of the CYP27B1 gene to MS in a Swedish population.

We replicate the previously identified association in the Australian and New-Zeeland population (ANZgene 2009). In a joint analysis the association of rs703842 to MS show an OR of 0.83 (0.79 -0.88 95%CI), reaching a significance 5.1 x 10 -11 lending further evidence to the involvement of CYP27B1 and vitamin D3 in MS.

The candidate region on chromosome 12q13-14 studied by the ANZgene consortium contains 17 genes. CYP27B1 is the most likely MS susceptibility candidate, given its importance in vitamin D3 activation and the important role of vitamin D3 in immunological functions. We have in this study confirmed the association between MS and the CYP27B1 gene. We cannot, however, exclude the possibility that our association to CYP27B1 is caused by linkage disequilibrium with some other marker in the 12q13-14 region such as polymorphisms in the MTTL1 gene. This study was a case-control study, although it has been performed in the fairly homogeneous Swedish population it is possible, although unlikely, that our observed association is due to population stratification between cases and controls. This could be corrected for by performing an association analysis in a family based material or by using genomic controls which unfortunately were not available for the included individuals [START_REF] Lange | Family-based samples can play an important role in genetic association studies[END_REF] .

Several studies show that MS patients have lower levels of 25-OHD 3 compared to controls 11 . In an Australian study, low levels of 25-OHD 3 could be linked to increased disability, likely caused by decreased sun exposure due to disability [START_REF] Van Der Mei | Vitamin D levels in people with multiple sclerosis and community controls in Tasmania, Australia[END_REF] . It has also been shown that there is an association between SNPs in the CYP27B1 region and 25(OH)D 3 levels [START_REF] Orton | Evidence for genetic regulation of vitamin D status in twins with multiple sclerosis[END_REF] .

Lower levels of 25-OHD 3 , and, 1,25(OH) 2 D 3 have also been seen in type 1 diabetes patients, and they also had a lower expression of CYP27B1 mRNA compared to controls. This reduction in expression could be linked to the GG variant of the rs10877012 polymorphism [START_REF] Ramos-Lopez | CYP2R1-, CYP27B1-and CYP24-mRNA expression in German type 1 diabetes patients[END_REF] . In our own material there was a significant difference in the number of GG homozygotes between cases and controls (cases frequency: 0.46, controls frequency: 0.42, p = 0.02). We would then expect a similar result in terms of CYP27B1 expression in our own material. One of the important immune genes regulated by vitamin D is the HLA class II locus, where there is a vitamin D receptor response element in the HLA-DRB1 promoter region, and highly conserved in the HLA-DRB1*1501 haplotype, which is the most important risk gene in MS. It was also shown that vitamin D3 had an effect on HLA-DRB1*1501 expression in vitro. However, the exact mechanism for the interaction between vitamin D3 and HLA and in which tissues or cells it takes place remains unknown [START_REF] Ramagopalan | Expression of the multiple sclerosis-associated MHC class II Allele HLA-DRB1*1501 is regulated by vitamin D[END_REF] .

Lower exposure to sunlight and low expression of CYP27B1 mRna will likely influence the amount of available active vitamin D3 in the body. In individuals with other genetic risk factor(s) and possibly exposed to other environmental factor(s), this could, through the effects of vitamin D3 on the immune system, lead to the development of MS or other autoimmune disease.

The results from studies on type 1 diabetes and CYP27B1 genotype and expression levels show that CYP27B1 expression could play a role in autoimmune disease. This should be replicated in MS patients, as well as studying the relationship between genotype and 25-OHD 3 levels. It will also be of value to study possible genetic interactions between CYP27B1, VDR and the HLA-complex, in particular HLA-DRB1*1501.

TABLES

  and rs703842 have been associated to 25(OH)D 3 levels in Canadian twins where at least one of the twins suffered from MS 10 . Vitamin D3 is naturally produced in the skin after UV B radiation, but can also be supplied in the diet. The enzyme 25-hydroxy-vitamin D-1 alpha hydroxylase, coded by the gene CYP27B1, hydroxylates the inactive form of vitamin D3, 25-hydroxyvitamin D3, into its bioactive form, 1,25-dihydroxyvitamin D3. This means that the final level of active vitamin D3 is dependent on both environmental factors, such as sun exposure and diet, and genetic factors such as the CYP27B1 gene. Bioactive vitamin D3 can bind to the vitamin D receptor (VDR) and has a regulatory role in calcium metabolism, as well as in immune functions. Vitamin D3 stimulation leads to decreased expression of MHC class II molecules and costimulators in B-cells, decreased proliferation, differentiation and cytokine production and immune response in T-cells 11,12 . A theory is that the production of CYP27B1, and activation of vitamin D3 by T-cells and DCs in extra-lymphoid tissues, might increase the T-cell

Swedish Cohort 1 :

 1 Swedish cohort 1 consisted of 1013 subjects (727 female) and 1215 blood donor controls (768 female). Patients were recruited from Danderyds Hospital and Karolinska University Hospital in Huddinge and in Solna, in the Stockholm County. Controls were blood donors that visited any of three blood donation facilities in the Stockholm area in 2001 and 2004/2005. Blood was consecutively sampled and re-sampling avoided by collection under a period of three month (under which blood donors are advised not to donate a second time) in 2001 or by explicitly asking if sample had previously been obtained (in 2004 and 2005). Controls were not screened for MS. Information about the study, along with questions regarding previous participation and ethnic origin of parents, was presented to donors both orally and in written form 16 . Swedish Cohort 2: These cases and controls are part of an ongoing population based casecontrol study called EIMS (Epidemiological Investigations in Multiple Sclerosis), described previously 17 . 664 patients (472 females, 1 unknown sex) and 544 controls (406 females) were available for genotyping. Patients and controls are recruited from all over Sweden. Controls are matched for age, sex, and residential location. Swedish Cohort 3: Patients (n=481, 347 female) recruited at Karolinska University Hospital Solna and from clinics all over Sweden. In total, 2158 cases (1546 females) and 1759 controls (1174 females) were included and available for genotyping. All cases fulfilled the MacDonald criteria. Possible overlaps between the cohorts were excluded using the national personal identification number for cases. All participants were of Scandinavian ancestry. Oral or written informed consent was obtained from all participants. The Ethical Committees at Karolinska University Hospital Huddinge and Karolinska University Hospital Solna approved the study. DNA extraction DNA was extracted from whole blood using standard procedures. DNA concentrations were measured using Picogreen TM fluorescence or with a ND-1000 Spectrophotometer (NanoDrop Technologies Inc, Wilmington, DE, USA). SNP selection Sundqvist et al. Confirmation of association between Multiple Sclerosis and CYP27B1

  Sundqvist et al. Confirmation of association between Multiple Sclerosis and CYP27B1

  Sundqvist et al. Confirmation of association between Multiple Sclerosis and CYP27B1

  Sundqvist et al. Confirmation of association between Multiple Sclerosis and CYP27B1

Table 1 :

 1 Summary of the SNPs genotyped and imputed in the Swedish material Total number of cases and controls genotyped, the minor allele frequency for each group and the number of individuals carrying the minor allele *NCBI build 36.3, MT-MS = MALDI-TOF MS.

					Alleles			Cases			Controls		HWE (p)	Association, allelic test
	SNP	Alt.name	Position* Minor Major	Method	Total N MAF	No. Total N MAF	No.	Controls	P-value	OR	95 % CI
	rs1048691		56439215	T	C	MT-MS	1938	0.222 760	1722	0.226 699	0.21	NS
	rs4646537		56443548	G	T	MT-MS	2072	0.038 161	1723	0.041 141	0.36	NS
	rs4646536 +2838T>C	56444255	G	A	Taqman/ MT-MS	2064	0.324 1118	1733	0.352 1008	0.79	0.01	0.88 0.80 -0.97
	rs8176345		56444825	T	C	MT-MS	2102	0.029 121	1751	0.030 101	0.02	NS
	rs2307096		56445011		C	MT-MS	2040	0.00		1708	0.00		1.00	NA
	rs10877012 -1260C>A	56448352	T	G	Taqman	1973	0.326 1075	1682	0.354 982	0.92	0.01	0.88 0.80 -0.97
	rs10877015		56454055	G	A	MT-MS	2086	0.321 1129	1741	0.353 1016	0.68	3.5x10 -3 0.87 0.79 -0.95
	rs703842		56449006	G	A	Imputed	2158	0.330 1209	1759	0.355 1037	0.32	0.02	0.89 0.81 -0.98

Sundqvist et al. Confirmation of association between Multiple Sclerosis and CYP27B1

Table 2 :

 2 The different study populationsThe three different study populations used in the combined analysis. Partially adapted from ANZgene. 2009. Sundqvist et al. Confirmation of association between Multiple Sclerosis and CYP27B1 Supplementary Table 1. Allele and haplotype frequencies in different Swedish cohorts

	Study	Cases	Controls Genotyped/Imputed
	ANZgene GWAS	1618	3413	Genotyped
	ANZgene replication	2256	2310	Genotyped
	Sweden	2158	1759	Imputed
	Total N	6032	7482

Acknowledgments

This study was supported financially by Neuropromise (LSHM-CT-2005-018637), Swedish Research Council, Swedish Foundation for the neurologically Disabled, the Söderberg foundation, Bibbi and Niels Jensens foundation, Swedish Council for Working life and Social Sundqvist et al. Confirmation of association between Multiple Sclerosis and CYP27B1 Research, the Swedish Associations for Persons with Neurological Disabilities, and the Montel Williams foundation.

Total number of cases and controls genotyped in each of the Swedish datasets. For each SNP and haplotype data over total samples genotyped, the minor allele frequency for cases and controls, p-values. There were no significant differences between the three groups.

Titles and legends to figures

Meta-analysis of the two ANZgene cohorts combined with the Swedish cohorts, with the allele count for cases and controls, and the odds ratio for each group. The combined odds ratio is 0.83 (0.79 -0.88 95%CI).


Maria Clara Bonaglia
email: clara.bonaglia@bp.lnf.

Susan Marelli

Francesca Novara

Simona Commodaro

Renato Borgatti

Grazia Minardo

Luigi Memo

Elisabeth Mangold

Silvana Beri

Claudio Zucca

Daniele Brambilla

Massimo Molteni

Roberto Giorda

Ruthild G Weber

Orsetta Zuffardi

Maria Clara Bonaglia

Genotype-phenotype relationship in three cases with overlapping 19p13.12 microdeletions

Keywords: 19p13.12 microdeletion, mental retardation, hearing loss Running title: 19p13.12 microdeletion

We describe the detailed clinical and molecular characterization of three patients (aged 7, 8 4/12 and 31 years) with overlapping microdeletions in 19p13.12, extending to 19p13.13 in two cases. The patients share the following clinical features with a recently reported 10-year-old girl with a 19p13.12 microdeletion (Jensen et al., 2009): mental retardation (MR), psychomotor and language delay, hearing impairment, brachycephaly, anteverted nares and ear malformations. All patients share a 359 kb deleted region in 19p13.12 harboring six genes (LPHN1, DDX39, CD97, PKN1, PTGER1, and GIPC1), several of which may be MR candidates because of their function and expression pattern. LPHN1 and PKN1 are the most appealing; LPHN1 for its interaction with Shank family proteins, and PKN1 because it is involved in a variety of functions in neurons, including cytoskeletal organization. Haploinsufficiency of GIPC1 may contribute to hearing impairment for its interaction with myosin VI. A behavioral phenotype was observed in all three patients; it was characterized by overactive disorder associated with MR and stereotyped movements (ICD10) in one patient and hyperactivity in the other two. Since Ptger1-null mice show behavioral inhibition and impulsive aggression with defective social interaction, PTGER1 haploinsufficiency may be responsible for the behavioral traits observed in these patients.

INTRODUCTION

De novo microdeletions detected by genome-wide array analysis at 19p13 have been reported in few patients, [START_REF] Engels | DNA microarray analysis identifies candidate regions and genes in unexplained mental retardation[END_REF][START_REF] Jensen | A novel chromosome 19p13.12 deletion in a child with multiple congenital anomalies[END_REF][START_REF] Aten | Split hand-foot malformation, tetralogy of Fallot, mental retardation and a 1 Mb 19p deletion-evidence for further heterogeneity?[END_REF][START_REF] Lysy | A new case of syndromic craniosynostosis with cryptic 19p13.2-p13.13 deletion[END_REF][START_REF] Auvin | Array-CGH detection of a de novo 0.7-Mb deletion in 19p13.13 including CACNA1A associated with mental retardation and epilepsy with infantile spasms[END_REF] all of whom had malformations and mental retardation. We present the detailed clinical and molecular description of two new cases (Cases 1 and 3) with partially overlapping 19p13 microdeletions identified by investigating patients with syndromic mental retardation using array comparative genomic hybridization (aCGH). We also re-analyzed at higher resolution and obtained the clinical follow-up of one published case 1 (Case 2) with a cryptic 19p13.12 deletion totally overlapping that of Case 1. The shortest region of overlap (SRO) between the three cases is 359 kb in size and contains candidate genes potentially responsible for their common phenotypic features (psychomotor, mental, language delay and sensorineural hearing loss). Genes outside the SRO are also discussed.

MATERIALS AND METHODS

Patients

All patients are described in detail in the section below. The clinical features of our subjects and of patients with overlapping 19p13.12 deletions reported by Jensen, [START_REF] Jensen | A novel chromosome 19p13.12 deletion in a child with multiple congenital anomalies[END_REF] Lysy, [START_REF] Lysy | A new case of syndromic craniosynostosis with cryptic 19p13.2-p13.13 deletion[END_REF] and Auvin [START_REF] Auvin | Array-CGH detection of a de novo 0.7-Mb deletion in 19p13.13 including CACNA1A associated with mental retardation and epilepsy with infantile spasms[END_REF] are summarized in Table 1.

Case 1

The proband is a male, the second child of healthy non-consanguineous parents aged 31 (mother) and 36 years (father) at his birth (Figure 1). Delivery was at 36 weeks of gestation by Caesarean section. Birth weight was 2400 g (3-10 th centile), and OFC was 32 cm (3-10 th centile). Length and APGAR scores were not available. At birth, some dysmorphic features were noted: malformed ears, submucous cleft palate, bilateral R1 cutaneous syndactyly of fingers II-III and toes, hypospadias. A standard karyotype was performed with normal results.

Early motor milestones were severely delayed: he acquired head control at 2 years and was able to walk without support at 3 years. He showed moderate mental retardation and language delay. Hyper-activity was also observed. [START_REF] Jerger | Clinical experience with impedence audiometry[END_REF] Bilateral over-the-ear hearing aids were positioned. Over follow up period of 4 years, the situation has remained stable. Scoliosis was also present at this age.

At the patient's last clinical evaluation (31 years), the following features were observed: short stature (height 150 cm; -3 SD), overweight (83 kg: BMI of 36,4 kg/m 2 ; +3 SD), and a relative small head circumference (OFC 53.5 cm; -1.3 SD); generalized hypertrichosis; nystagmus; brachycephaly, synophrys, symmetrically arranged, vaguely outlined nodules and partly confluent plaques accompanied by a fuzzy erythema on the forehead, arched eyebrows, mild blepharophimosis, small/ thin nasal root, anteverted nares, anteverted ears with thin helices, short neck (Figure 1D,E), brachydactyly (Supplementary Figure 1); bilateral cutaneous syndactyly of fingers II-III-IV; bilateral cutaneous syndactyly of toes II-III. Neurological examination revealed no focal signs.

He walked with small steps. Subclinical hypothyroidism and hyperlipidemia were also present. By abdominal ultrasound, mild hepatic steatosis was diagnosed; echocardiography confirmed mild aortic and mitral valve regurgitation.

Case 2

This patient was previously included in a mental retardation cohort study (patient 11/3); [START_REF] Engels | DNA microarray analysis identifies candidate regions and genes in unexplained mental retardation[END_REF] we provide here a more detailed clinical and molecular characterization. This female patient is the only child of unrelated parents, both 26 years old when she was born. Her father was known to suffer from epilepsy since childhood.

Pregnancy was initially uneventful. After week 21, IUGR was noted on repeated ultrasound examinations. Delivery took place at 32+1 weeks by Caesarean section.

Birth weight was in the lower normal range (between 3 rd and 10 th centile), length was at R1Q5 the 3 rd centile and microcephaly was noted (head circumference below the 3 rd centile; -2.4 SD). Shortly after birth, a mandibular neonatal tooth and a skin tag located on the tragus of the right ear were noticed.

Within the first weeks of life, weight and length dropped below the 3 rd centile. At the age of 1 month, a persisting ductus Botalli was surgically treated. Motor development was moderately delayed (unsupported sitting at 15 months and first attempts to crawl at 16 months) and there was no speech.

Abnormal results of repeated hearing tests raised the suspicion of bilateral sensorineural hearing loss. She developed muscular hypotonia and gastroesophageal reflux, and a persistent foramen ovale was noted. Cranial MRI scan, EEG and eye examination at the age of 11 months revealed no abnormalities. No evidence for metabolic disorders was found.

At the age of 16 months, the patient appeared slightly hypotonic and brachycephaly was observed. Facial dysmorphisms (synophrys, almond-shaped eyes with a bilateral epicanthic fold, long lashes, mild ptosis, flattened nasal bridge, anteverted nostrils, long philtrum, small mouth with thin upper lip) were noticed. Ears were small and posteriorly rotated and the neck was short. An atypical Simian crease was noted on her right hand.

At the age of 8 4/12 years the patient had developed hyperopia and moderate mental retardation with marked hyperactivity. Microcephaly (-3,2 SD) and brachycephaly were observed, height and weight were within the normal range. In addition to the facial dysmorphisms described earlier, her face now appeared round and slightly flattened, and a small nasal root and thin lower lip were noted. Philtrum and neck now had a normal length. The two lateral incisors were missing in her upper jaw.

Case 3

The proband is a male, the second child of healthy, non-consanguineous parents. There is no family history of malformations or mental retardation.

He was born by spontaneous delivery at 41 weeks of gestational age after an uncomplicated pregnancy. APGAR scores were 9/10/10. His birth weight was 2740 g (< 3 rd centile), length 47 cm (< 3 rd centile) and head circumference 34 cm (10-25 th centile). At 3 days, the patient presented remarkable hypotonia and recurrent apneas accompanied by bradycardia without desaturation were observed. He was therefore admitted to a neonatal intensive care unit, where a cerebral ultrasound showed a right choroid plexus cyst and subependymal hemorrhage. ECG, echocardiography and fundoscopy were also performed with normal results. At 2 and 5 years, brain MRI scans were carried out revealing no morphological abnormalities. EMG showed normal conductance in the muscles tested and in peripheral nerves.

At the age of 5 years and 1 month he showed a focal epileptic syndrome. He sporadically presented with focal left-sided seizures. Because of the extremely low frequency of the seizures, chronic anti-epileptic treatment was never started. EEG recordings showed epileptiform abnormalities over the right central areas.

The following distinctive features were observed: brachycephaly, small mouth with thin upper lip, long philtrum, anteverted ears, irregular teeth, bilateral epicanthic folds, brachydactyly, pectus excavatum, scoliosis, muscle hypotonia and cryptorchidism.

Severe speech and neuromotor delay were also noted. His behavioral phenotype was characterized by hyperactivity.

At last evaluation (7 yrs old)(Figure 2), the patient showed moderate-severe speech and neuromotor retardation. His measurements were all within normal range. Bilateral conductive hearing loss was present.

MOLECULAR STUDIES

Routine karyotyping by G-banding analysis at 400-550 bands was performed using standard procedures. Array-CGH was performed with the Agilent oligo-platform at a resolution of ~100 Kb (Agilent kit 44B) in all three cases. The procedures for DNA digestion, labeling, and hybridization were executed according to the manufacturer's instructions. Array slides were analyzed by the Agilent scanner (G2505C) with Feature Extraction software (v10.1.1.1). Graphical overviews were obtained using the Agilent DNA analytics software (v4.0.81).

Genotyping of polymorphic loci, quantitative PCR (qPCR), long-range PCR (LR-PCR)

and DNA sequencing were performed as described. [START_REF] Bonaglia | A familial inverted duplication/deletion of 2p25.1-25.3 provides new clues on the genesis of inverted duplications[END_REF] The map used to determine the probes' genomic positions was Genome Browser Assembly hg18

(http://genome.ucsc.edu). Based on the aCGH characterization of the deletions, we identified the regions between last non-deleted and first deleted probes on each side of the breakpoint. For Case 1, we selected four approximately evenly spaced primers on non-repeated portions of the distal breakpoint region and three primers on the proximal region, and performed long-range PCRs with all possible distal/proximal primer combinations. One combination generated a 4.5 kb fragment that was entirely sequenced. For Case 2, we selected three qPCR primer pairs in non-repeated portions of the distal breakpoint region, two pairs in the proximal region, and narrowed both regions down to 3-4 kb. Then we performed long-range PCR and generated a 5 kb fragment that was entirely sequenced. For Case 3, we selected four evenly spaced qPCR primer pairs in the distal breakpoint region and eight pairs in the proximal region. By qPCR, we narrowed both regions down to 6-8 kb, then selected three long-range PCR primers on each side and tested all combinations. The shortest resulting fragment (2.5

Kb) was again entirely sequenced.

RESULTS

Molecular analysis

In the absence of an etiological diagnosis, aCGH was performed to detect submicroscopic imbalances in Cases 1 and 3 and to redefine the chromosomal breakpoints in the case reported by Engels 1 (Case 2) with the same array platform (Agilent, kit 44B) (Figure 3B).

The results were:

Case 1: deletion of 1.9 Mb in chromosomal band 19p13.12 with breakpoints at 14.119 - Typing of informative polymorphic chromosome 19 markers D19S432 and D19S558 in the deleted interval in Cases 1, 3 and their parents demonstrated that in both patients the deletion was of maternal origin. For Case 2, parental samples were not available to assess the origin of the deletion.

Breakpoint cloning and sequencing

The breakpoints characterized by aCGH were further defined by qPCR, amplified by LR-PCR, and sequenced in all three cases. The positions and sequences of all junctions are shown in Figure 3C.

In Case 1, a 3 bp ATC microduplication, evidence of microhomology-related nonhomologous end joining (NHEJ), is present at the junction. The distal breakpoint falls in the 3'-end of the LPHN1 gene, while the proximal breakpoint is in intron 2 of TPM4.

In Cases 2 and 3, DNA sequence analysis revealed that all breakpoints were located in Alu repeat elements and that both deletions originated by Alu-mediated homologous recombination. In Case 3, the distal breakpoint is in intron 3 of the SYCE2 gene, while the proximal breakpoint is located between GIPC1 and DNAJB1. In Case 2, the distal breakpoint is in intron 2 of the RFX1 gene, the proximal breakpoint in intron 7 of the TPM4 gene. The proximal breakpoints of Cases 1 and 2 are only 15 kb apart. In Cases 1 and 2, the rearrangement does not generate any functional fusion gene because the genes involved are in opposite orientation. None of the breakpoints is located within or near segmental duplications.

DISCUSSION

In this study, we report three patients with partially overlapping microdeletions in 19p13.12. Case 1 was analyzed using aCGH because of his abnormal behavior with catastrophic conduct characterized by auto-and hetero-aggressivity.

Case 2 was briefly reported by Engels 1 and belonged to a cohort of patients selected for aCGH because of unexplained mental retardation. Case 3 was studied for mental retardation and difficult-to-manage hyper-activity.

A comparison of the clinical findings in the three cases is hampered by the fact that patients' ages at the times of last evaluation were very different (i.e. 7 years, 8 4\12 years and 31 years) and that the oldest patient's history is incomplete for the first months and years of his life. Changes of phenotype with age are well documented in patients with chromosomal imbalances. [START_REF] Van Buggenhout | Cri du chat syndrome: changing phenotype in older patients[END_REF] To increase the numbers of patients and to bridge the age gap, we compared the phenotypes of our patients with the recently reported case 2 of a 10-year-old girl carrying a 2.52 Mb deletion in 19p13.12-p13.13 overlapping the deleted region in our patients (Figure 3A).

The clinical features common to our patients and to the case reported by Jensen 2 are moderate to severe psychomotor delay, language delay, hearing loss, and facial dysmorphisms including brachycephaly, anteverted nares, as well as ear malformations.

Mild congenital cardiac anomalies and/or rhythm disturbance were observed at birth in all cases, and resolved spontaneously in the first years of life, with the exception of mild aortic and mitral valve incompetence that was still present at the age of 31 years in Case 1 (Table 1).

The four cases share a 358,802 bp SRO where six annotated genes lie:

LPHN1/CIRL1, CD97, DDX39, PKN1, PTGER1, and GIPC1 (Figure 3A).

Two additional recently published cases carry 664 Kb 5 and 3 Mb 4 deletions of 19p13.13-p13.2 partially overlapping the distal portion (including the SYCE2, NFIX, and CACNA1A genes) of the deletion detected in our Case 3 (Figure 3A). There is no overlap between these deletions and those in our Cases 1 and 2.

LPHN1/CIRL1 encodes the latrophilin-1 precursor/calcium-independent receptor for alpha-latrotoxin, a member of the latrophilin subfamily of G-protein coupled receptors (GPCR) and may function in both cell adhesion and signal transduction.

Interestingly, LPHN1 interacts with with the PSD-95/discs large/ZO-1 (PDZ) domain of the multidomain protein family ProSAP/SSTRIP/Shank. 9 Shank1 knockout mice show smaller dendritic spines in CA1 pyramidal neurons and weaker synaptic transmission, along with altered learning and memory. 10 Haploinsufficiency or point mutations of SHANK3, another member of the SHANK family, results in language delay and/or social communication disorders. 11,12 Deletion of LPHN1 may be potentially responsible for the cognitive and language delay and might contribute to the behavioural phenotype observed in our cases.

The GIPC1 gene, also named synectin (MIM 605072), encodes a scaffolding protein that regulates cell surface receptor expression and trafficking 13 and may be involved in G protein-linked signalling. The GIPC1 gene is annotated in the G2C cognition database (Human Gene ID: G00001513; Mouse Gene ID: G00000264; http://www.genes2cognition.org). Haploinsufficiency of GIPC1 may also contribute to the mental and language delay in our patients. In addition, the GIPC1 gene may be a good candidate for the hearing impairment observed in all four patients, since its product binds to myosin VI, 14 encoded by the MYO6 gene. The C442Y missence mutation in this gene may compromise myosin VI function in a dominant-negative manner and cause autosomal dominant nonsyndromic hearing loss. 15 However, the patient described by Lysy 4 had bilateral hearing loss, despite the fact that his deletion did not extend to the GIPC1 gene. Furthermore, no hearing loss was described in the Gipc1 knock-out mouse. 16,[START_REF] Naccache | Binding of internalized receptors to the PDZ domain of GIPC/synectin recruits myosin VI to endocytic vesicles[END_REF] Thus, the role of GIPC1 with respect to hearing impairment still remains to be clarified.

PKN1 (MIM 601032) codes for a protein belonging to the protein kinase C superfamily and is implicated in a variety of functions in neurons, including R1Q6 cytoskeleton organisation. [START_REF] Mukai | The structure and function of PKN, a protein kinase having a catalytic domain homologous to that of PKC[END_REF] The recent demonstration that deregulation of PKN1 activity disrupts neurofilament organisation and axonal transport [START_REF] Manser | Deregulation of PKN1 activity disrupts neurofilament organisation and axonal transport[END_REF] suggests that its haploinsufficiency may have an impact on the mental and language delay in our patients.

The PTGER1 gene (MIM 176802) codes for a protein belonging to the G protein-coupled receptor family. Ptger1-null mice display behavioral inhibition, manifested as impulsive aggression with defective social interaction, impaired cliff avoidance, and an exaggerated acoustic startle response when under social or environmental stress. [START_REF] Matsuoka | Prostaglandin E receptor EP1 controls impulsive behavior under stress[END_REF] Haploinsufficiency of this gene might be responsible for the severe behavioral traits in Case 1 and the hyper-activity in Case 2 and Case 3.

Based on the current knowledge, deletion of CD97 (MIM 601211) and DDX39

should not have any impact on patients' phenotype.

Several recent papers have shown that deletion or duplication of the region flanking a gene might also be disease causing. [START_REF] Benko | Highly conserved non-coding elements on either side of SOX9 associated with Pierre Robin sequence[END_REF][START_REF] Dathe | Duplications involving a conserved regulatory element downstream of BMP2 are associated with brachydactyly type A2[END_REF][START_REF] Kurth | Duplications of noncoding elements 5' of SOX9 are associated with brachydactyly-anonychia[END_REF] Thus, we also considered genes flanking the SRO as potentially responsible for the abnormal phenotypes.

On the proximal side (Figure 3A), SLC1A6 (MIM 600637) codes for a protein that transports L-glutamate, L-and D-aspartate, and seems to act as a symport by cotransporting sodium. It is expressed in the brain and in cell bodies of Purkinje cells.

Association studies of polymorphisms in glutamate transporter genes demonstrated that SLC1A6 is a susceptibility locus for schizophrenia. [START_REF] Deng | Association study of polymorphisms in the glutamate transporter genes SLC1A1, SLC1A3, and SLC1A6 with schizophrenia[END_REF] SLC1A6 is deleted in Cases 1, 2, and in Jensen's case. [START_REF] Jensen | A novel chromosome 19p13.12 deletion in a child with multiple congenital anomalies[END_REF] Its haploinsufficiency could be responsible for cognitive and language delay in all subjects and may contribute to the severe behavioral phenotype in Case 1.

On the distal side (Figure 3A), PRKACA (MIM 601639) codes for a catalytic subunit of the cAMP-dependent protein kinase that is required for long-term potentiation (LTP) in neonatal tissue. [START_REF] Yasuda | A developmental switch in the signaling cascades for LTP induction[END_REF] LTP is thought to be critically involved not only in learning and memory, but also in neural circuit formation and refinement. We suggest that haploinsufficiency of PRKACA may be responsible for the cognitive and language delay in Cases 2, 3, in the subject reported by Jensen, 2 and possibly also in Case 1 if a functionally null allele for PRKACA was created by a position effect or by disruption of regulatory elements.

Mutations in the CACNA1A gene (MIM 601011) have been reported in association with epilepsy and status epilepticus [START_REF] Jouvenceau | Human epilepsy associated with dysfunction of the brain P/Q-type calcium channel[END_REF][START_REF] Beauvais | New CACNA1A gene mutation in a case of familial hemiplegic migraine with status epilepticus[END_REF][START_REF] Kors | Childhood epilepsy,familial hemiplegic migraine, cerebellar ataxia, and a new CACNA1A mutation[END_REF] and its haploinsufficiency has been suggested to be responsible for epilepsy and infantile spasms in Auvin's case, [START_REF] Auvin | Array-CGH detection of a de novo 0.7-Mb deletion in 19p13.13 including CACNA1A associated with mental retardation and epilepsy with infantile spasms[END_REF] as well as for the abnormal electroencephalography in Lysy's case. [START_REF] Lysy | A new case of syndromic craniosynostosis with cryptic 19p13.2-p13.13 deletion[END_REF] CACNA1A deletion is most likely responsible for the spontaneous focal seizures in Case 3, while a positional effect concomitant to antipsychotic treatment might have evoked the epileptic seizure observed in Case 1.

NFIX (MIM 164005) plays an essential role in the development of the brain and skeleton. [START_REF] Driller | Nuclear factor I X deficiency causes brain malformation and severe skeletal defects[END_REF] Nfix-deficient mice exhibit enlargement of the lateral and third brain ventricles and partial agenesis of the corpus callosum; additionally, they display kyphotic deformation of the spine and impaired endochondral ossification in the vertebrae and femur. [START_REF] Driller | Nuclear factor I X deficiency causes brain malformation and severe skeletal defects[END_REF] Lysy's case showed kyphosis, artogryphosis of lower limb craniosynostosys and moderate ventriculomegaly; [START_REF] Lysy | A new case of syndromic craniosynostosis with cryptic 19p13.2-p13.13 deletion[END_REF] Auvin 5 reported advanced bone age.

Since our Case 3 does not show any of these malformations, the phenotype associated with NFIX haploinsufficiency may occur with variable penetrance.

In conclusion, we described two new cases of 19p13.12-p13.13 deletions, refined the breakpoints of another, [START_REF] Engels | DNA microarray analysis identifies candidate regions and genes in unexplained mental retardation[END_REF] and compared the data with three published patients. [START_REF] Jensen | A novel chromosome 19p13.12 deletion in a child with multiple congenital anomalies[END_REF][START_REF] Lysy | A new case of syndromic craniosynostosis with cryptic 19p13.2-p13.13 deletion[END_REF][START_REF] Auvin | Array-CGH detection of a de novo 0.7-Mb deletion in 19p13.13 including CACNA1A associated with mental retardation and epilepsy with infantile spasms[END_REF] We found that patients with a 358,802 bp common deleted portion at 19p13.12 are characterised by psychomotor and language delay, mental retardation, bilateral sensorineural and/or conductive hearing loss brachycephaly, anteverted nares, and ear malformations, as well as mild congenital cardiac anomalies at birth. Patients with deletion of CACNA1A show epilepsy or epileptiform abnormalities on EEG recording.

Several genes within the deletion region may be associated with mental retardation because of their function and expression pattern. The most appealing among them are LPHN1, for its interaction with the SHANK proteins, and PKN1. The GPC1 gene may be a candidate for the hearing impairment observed in all four patients. Case 1 is the most peculiar subject, due to his difficult to manage aggressive behavioural phenotype, most pronounced after adolescence. In cases 2 and 3, hyperactivity was observed.

Haploinsufficiency of the PTGER1 gene may be responsible for these behavioural traits.

However, it is also possible that specific phenotypic traits are due to a multi-gene effect associated with several of the numerous genes included in each deletion.

Additional cases, with different ages and breakpoints, will help to define the phenotype and clarify the role of each deleted gene.

  At the age of 11 years, a diagnosis of praecox puberty was made. Cardiac ultrasound showed mild aortic and mitral valve regurgitation. Brain CT scans were normal. During adolescence, repetitive and stereotyped behavior worsened, with iterative questions and very limited interests. He developed catastrophic behavior as a response to minimal environmental changes, with auto-aggressive and sometimes hetero-aggressive acting out. A diagnosis of overactive disorder associated with mental retardation and stereotyped movements (ICD 10) was made and specific drug therapy (clomipramine, chlorpromazine and fluvoxamine ) was started with progressive improvement. At 24 years, the patient showed several generalized clonic seizures with falls after an increase of the fluvoxamine dosage. Despite its potential pro-convulsive effects, antipsychotic therapy could not be tapered and antiepileptic chronic treatment was started with sodium valproate. Complete seizure control was achieved with a follow-up period of 8 years. EEG recordings showed a global slowing of background activity without epileptiform abnormalities. At the age of 27 years, Auditory Brain Middle Latency Responses (ABR and MLR) and Behavioral Audiometry (VRA) exams revealed bilateral sensorineural hearing loss. The measurements obtained by pure tone audiometry (500-1000 Hz and 2000 Hz), were 65 dBHL (right ear) and 80 dBHL (left ear). Tympanometry results were normal in the right ear, whereas the left ear was classified as type B, according to Jerger's R1Q2 R1Q4 R1Q4 classification.

Figure 1

 1 Figure 1 Photographs of Case 1 at the age of six months (A), 8 years (B), 17 years (C);

Figure 2

 2 Figure 2 Photographs of Case 3 at the age of 7 years frontal (A, C) and lateral (B) view.

Figure 3

 3 Figure 3

Table 1

 1 Melchionda S, Ahituv N, Bisceglia L et al: MYO6, the human homologue of the gene responsible for deafness in Snell's waltzer mice, is mutated in autosomal dominant nonsyndromic hearing loss. Am J Hum Genet 2001; 69: 635-640. 16 Chittenden TW, Claes F, Lanahan AA et al: Selective regulation of arterial branching morphogenesis by synectin. Dev Cell 2006; 10: 783-795. Clinical and cytogenetic characteristics of patients with 19p13.12 deletions. *Case reported by Engels (case 11/03). 1 NR, not recorded; NT, not tested. ASD: atrial septal defect; VSD: ventricular septal defect. When a specific clinical feature had not been described in the original report the item was not scored.

	9 Kreienkamp HJ, Zitzer H, Gundelfinger ED, Richter D and Bockers TM The
	calcium-independent receptor for alpha-latrotoxin from human and rodent brains
	interacts with members of the ProSAP/SSTRIP/Shank family of multidomain
	proteins. J Biol Chem 2000; 275: 32387-32390.
	10 Hung AY, Futai K, Sala C et al: Smaller dendritic spines, weaker synaptic
	transmission, but enhanced spatial learning in mice lacking Shank1. J Neurosci
	2008; 28: 1697-1708.
	11 Bonaglia MC, Giorda R, Mani E et al: Identification of a recurrent breakpoint
	within the SHANK3 gene in the 22q13.3 deletion syndrome. J Med Genet 2006; 43:
	822-828.

12 Durand CM, Betancur C, Boeckers TM et al: Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are associated with autism spectrum disorders. Nat Genet 2007; 39: 25-27. 13 Lee NY, Ray B, How T and Blobe GC Endoglin promotes transforming growth factor beta-mediated Smad 1/5/8 signaling and inhibits endothelial cell migration through its association with GIPC. J Biol Chem 2008; 283: 32527-32533. 14 Aschenbrenner L, Lee T and Hasson T Myo6 facilitates the translocation of endocytic vesicles from cell peripheries. Mol Biol Cell 2003; 14: 2728-2743. 15

Acknowledgements

We thank the families for their cooperation. This work was supported by Italian Ministry of Health grant RF-AOM-2007-636538 to O.Z

including #5089, and 5090 in the SRO region, have been detected, at very high frequency, exclusively in one report using BAC-array analysis, [START_REF] Wong | A comprehensive analysis of common copy-number variations in the human genome[END_REF] and may therefore be somewhat unreliable. B, aligned aCGH profile details of the deletions; the deleted regions are shaded in grey. C, sequence alignment of the breakpoint junctions; the 3 bp microhomology in Case 1 and informative mismatching bases in Cases 2 and 3 are shown in bold.

Supplementary Figure Legends

Supplementary Figure 1 Photographs of the hands of Case 1: dorsal (top) and ventral (bottom) view.


Piranit Stefan Mundlos
email: stefan.mundlos@charite.de

Nik Kantaputra

Eva Klopocki

Bianca P Hennig

Cedric Le Caignec

Verayuth Praphanphoj

Mei Lan Kwee

Deborah J Shears

Bertrand Isidor

Piranit N Kantaputra

Cédric Le Caignec

Kantaputra type mesomelic dysplasia is associated with duplications of the locus on chromosome 2q

Keywords: HOXD, mesomelic dysplasia Kantaputra type, microduplication

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

Introduction

Kantaputra type mesomelic dysplasia (MDK, MIM 156232) is a rare dominantly inherited skeletal disease characterized by symmetric marked shortening of the upper and lower limbs 1,2 . The ulnae are very short, and the radii bowed. The distal humerus has a dumb bell shape, the hands are relatively normal but show progressive flexion contractures of the proximal interphalangeal joints. Carpal and tarsal synostoses are observed in some individuals. In the lower limbs, feet are fixed in plantar flexion with sole facing backward, causing "ballerina-like standing". The prominent distal fibula on the ventral aspect is a consistent feature and considered the "signature" finding of the syndrome. Calcaneus is small or missing. Small fibula and talus, and fibulo-calcaneal synostosis are characteristic features. Tibial bony knot articulates with the proximal end of the fibula. Since the original description several other cases have been described that show similar changes 3,4 . In an Italian family with a mesomelic dysplasia a balanced translocation t(2;8)(q31;p21) had previously been observed 5,6 . Based on the phenotypic similarities with MDK chromosome 2 and 8 were tested as candidate regions and a region on chromosome 2 (2q24-q32) spanning about 22.7 cM was identified to harbour the MDK gene 7 . Interestingly, this interval contains the HOXD cluster. However, sequencing of individual genes of the HOXD cluster revealed no mutation associated with MDK. We here describe genomic rearrangements including the entire HOXD cluster identified in the Thai family originally described by Kantaputra et al. 1,2 as the likely cause for MDK.

Methods and Materials

Genomic DNA samples were obtained from EDTA-blood. DNA of the nine living affected individuals and one unaffected individual (IV.23) from pedigree of the 1 2 publication of Kantaputra et al. 2 were available for genomic analysis by array CGH and qPCR. Additionally, we obtained DNA samples of the MDK affected individual II.1 from the publication of Shears et al. 3 and II.1 from the publication of Kwee et al. 4 .

Microarray-based comparative genomic hybridization (array CGH)

DNA of the affected individual III.26 2 , II.1 3 and II.1 4 were available for array-CGH analysis. Array CGH was performed using a whole genome oligonucleotide array (244K, Agilent Technologies, Santa Clara, CA). 244K image data were analyzed using Feature Extraction 9.5.3.1 and CGH Analytics 3.4.40 software (Agilent Technologies, Santa Clara, CA) with the following analysis settings: aberration algorithm ADM-2; threshold: 6.0; window size: 0.2 Mb; filter: 5probes, log2ratio = 0.29. The hybridization on a custom tiling array (AMADID# 023120; Agilent Technologies, Santa Clara, CA) which covers 3 Mb of the critical region at high density allowed a more precise determination of the duplication size and breakpoint positions and was performed exclusively with the Thai family member III.26 2 . All genomic positions given in the text are according to human genome version hg18.

Quantitative real-time PCR (qPCR)

Confirmation of the array CGH result of individual III.26 2 and analysis of further nine members (eight affected, one unaffected) of the Thai MDK family 2 to show the segregation was done by quantitative real-time PCR. Amplicons P1 and P3 are flanking the centromeric duplication and amplicons P4 and P6 are flanking the telomeric duplication. Amplicons P2 and P5 are located within the centromeric and telomeric duplication, respectively. Primer sequences can be obtained upon request. qPCR was performed on ABI Prism® 7900HT Sequence Detection System in a total volume of 12µl in each well containing 6µl of SYBR-Green PCR Master Mix (ABI SYBR® Green PCR Master Mix), 10ng of genomic DNA (10µl) and 1µl primers (0.2µmol each). Samples were run in triplicates in separate tubes to permit the quantification of the target sequences normalized to albumin (ALB). PCR conditions were according to manufacturer's protocol: initial denaturation step of 95°C for 8 minutes followed by 40 cycles with denaturation at 95°C for 15 seconds and a combined annealing/elongation step at 60°C for 1 minute. By using calibrator samples of normal control genomic DNA the gene copy number was estimated based on the ddCt method. In addition, we performed an identification of the individuals' genders calculating the coagulation factor VIII (F8, Xq28) relative to the two-copy-control ALB to assure its reliability.

Fluorescence in situ hybridization (FISH)

BAC clones RP4-745K6 and RP11-279N12 (obtained from the imagenes, Berlin, Germany) were fluorescently labeled using nick translation, and hybridized to metaphase spreads of the individual III.11's lymphocytes using standard procedures.

BAC clones were labeled with Spectrum Orange® (RP4-745K6, located in centromeric duplication) and Spectrum Green® (RP11-279N12, located in telomeric duplication).

Chromosomes were counterstained with DAPI. Image analysis was performed using the ISIS analysis system (Metasystems, Altlussheim, Germany).

5

Results

Since submicroscopic aberrations are a known cause for congenital limb malformations 8-10 we performed array CGH using a whole genome oligonucleotide array (244K, Agilent Technologies, Santa Clara, CA, USA) of one affected (III.26) from the MDK family initially described by Kantaputra 2 . Using this technology we identified two microduplications on chromosome 2 (2q31.1-q31.2) encompassing approx. 481 and 507 kb, respectively, separated by a segment of normal copy number. The centromeric duplication contained the entire HOXD locus plus EVX2 and MTX2, whereas the telomeric duplication contained the genes NEF2L2 and HNRNPA3. To determine the duplication sizes and the breakpoints in more detail, we designed a custom tiling array of the entire region of approx. 3 Mb. This technology confirmed both duplications in the affected MDK family member III.26 2 and positioned them at 176,506,567-176,980,136 (centromeric dup) and 177,342,561-177,854,004 (telomeric dup), respectively (Fig. 1A). A cryptic translocation or insertion of the duplicated regions into another chromosome was excluded by FISH analysis (data not shown).

By quantitative PCR analyses we were able to confirm the duplications and further narrow down their boundaries: 1) centromeric duplication, proximal breakpoint between 176,506,582 and 176,507,065 and distal breakpoint between 176,985,080 and 176,986,597; 2) telomeric duplication, proximal breakpoint between 177,342,108 and 177,342,602 and distal breakpoint between 177,856,721 and 177,857,706 (data not shown). The proximal breakpoint of the centromeric duplication was thus located within the gene KIAA1715 and the distal breakpoint ~221 kb distal of MTX2. The telomeric duplication started ~357.5 kb away from the centromeric duplication and ended in the non-coding region between the genes NFE2L2 and APGS. In addition, we identified a small amplification at the distal end of the centromeric duplication (chr2:176,973,781-176,980,910) (Fig. 1A). However, this region overlaps with a benign copy number variation (CNV; chr2:176,973,862-176,980,178) described by Kim et al. 11 and it therefore most likely not clinically relevant. Segregation of the duplications with the MDK phenotype was confirmed by qPCR analysis of further nine family members from the Thai family (eight affected, one unaffected). Amplicons P2 and P5 which localize within the duplications detected a normal copy number in the unaffected individual IV.23 (Fig. 1B). The unaffected individual is the daughter of the affected female III.26 tested by array CGH. In order to investigate other non-related individuals with MDK we tested two previously published cases by array CGH 3,4 . We were not able to identify a clinically relevant copy number change, not within the region on chromosome 2q, nor elsewhere in the genome indicating that MDK is a genetically heterogeneous condition..

Discussion

Homeobox genes, and the proteins they encode, the homeodomain proteins, play important roles in the developmental processes of many multicellular organisms. In mammals four such clusters exist that harbor a total of 49 genes. During development of the limbs HOX genes of the A and D clusters, and in particular those of the 5' end, i.e. HOXA10, -A11, -A13, HOXD9, -D10, -D11, -D12, -D13, are of particular importance 12 .

Overall they appear to systematically involve loss or reduction of skeletal elements and their function has accordingly been interpreted to specify individual segments of the limb in a proximo-distal fashion.

In humans, mutations in HOX genes results in a number of limb malformations including synpolydactyly (HOXD13), and hand-foot-genital syndrome (HOXA13) 13 .

Larger deletions of the entire HOXD-cluster and a smaller deletion removing just the HOXD9-HOXD13 and EVX2 genes were reported to result in mild limb malformations including fifth finger clinodactyly, variable cutanous syndactyly of the toes, hypoplastic middle phalanges of the feet and synpolydactyly 14,15 . This phenotype is similar to the mild synpolydactyly phenotype commonly found in carriers of specific HOXD13 mutations indicating that even deletions of the entire HOXD cluster need not to produce major limb defects 13 . Several chromosomal translocations located within the vicinity of the HOXD cluster have been described that result in severe phenotypes (Fig. 1). Two rearrangement breakpoints located on the telomeric side of the HOXD cluster were described to result in proximal malformations of the limbs 16 . In one individual a symmetrical limb phenotype consisting of ulnar aplasia, radial shortening, and absence of 3rd to 5th rays was described in association with the translocation t(2;10)(q31.1;q23.33). The breakpoint was localized approx. 950 kb away from the 3' end of the HOXD cluster. In the other case a paracentric inversion [inv(2)(p15q31)] was associated with hypoplastic ulnae, radii and fibulae and small tibiae. This phenotype is somewhat reminiscent of the changes seen in MDK. The breakpoint in this case was closer to the cluster with a distance of approx. 500 kb from HOXD1 16 .

Furthermore, a refinement of the translocation t(2;8) of the patient with mesomelic shortening originally described by Ventruto and colleagues 5 located this breakpoint 56 kb away from the 3' end of the HOXD cluster 6,17 (for overview see Fig. 1C).

The above described breakpoints localize within or close to the here described duplications. Several other genes besides the HOXD cluster are present in this region that are potential candidates for MDK. The centromeric duplication contains parts of KIAA1715, EVX2 and MTX2. KIAA1715, also known as Lunapark (LNP), is expressed in the developing digits but does not seem to have an important function during digit development 18 . EVX2, located next to HOXD13, is also expressed in the digits.

Inactivation of Evx2 in the mouse results in weak skeletal alterations of the digits 19 . An additional copy of EVX2 may thus have an effect on limb development, but the main effect of EVX2 appears to be in the autopod, the region of the limb that shows little changes in MDK. MTX2 (MIM 608555) encodes for Metataxin, a protein that localizes to the outer mitochondrial membrane. It is not expressed in the developing limb 17 and as such unlikely to contribute to the phenotype. The telomeric duplication neighbors the AGPS gene, a member of the FAD-binding oxidoreductase/transferase type 4 family involved in long chain fatty acid metabolism. Mutations in this gene have been associated with rhizomelic chondrodysplasia punctata, type 3 (MIM 600121) and Zellweger syndrome (MIM 214100). Located within the duplicated region are HNRNPA3 and NFE2L2 (MIM 600492). The latter encodes a transcription activator that is important for the coordinated up-regulation of genes in response to oxidative stress.

HNRNPA3 plays a role in cytoplasmic trafficking of RNA and may be involved in pre-mRNA splicing. In summary, it is unlikely that either of these genes is involved in the pathogenesis of MDK.

Several lines of evidence suggest, however, that HOXD genes are the most likely candidates for the MDK phenotype. HOX genes show specific proximo-distal expression patterns that specify the future skeletal elements. HOXD9 and -D10, for example, are expressed in the stylopod, the future humerus, HOXD11 and HOXA11 in the zeugopod, the future radius/ulna and HOXD13, HOXD12, and HOXA13 in distal limb (autopod). Inactivation of paralogous genes results in severe shortening of the corresponding elements. A knock-out of Hoxd11 and Hoxa11, for example, results in a severe reduction deficiency of radius and ulna, similar to the MDK phenotype 20,21 . MDK-like malformations are also present in the mouse mutant ulnaless (Ul) which is a semidominant x-ray induced mutant with very short zeugopods with an almost complete absence of the ulna. Gene expression studies have shown that Ul embryos show an ectopic expression of Hoxd13 and Hoxd12 in the proximal limb (zeugopod) and a reduction of Hoxd13, Hoxd12 and Hoxd11 in the autopod 22,23 . The skeletal reductions of the Ul mutant were interpreted as a consequence of posterior prevalence, whereby proximal misexpression of Hoxd13 and Hoxd12 results in the transcriptional and/or functional inactivation of the more anterior, i.e. Hox group 11 genes. The hypothesis of misregulated Hoxd13 expression was supported by the finding that Ul was caused by a balanced paracentric inversion of approx. 770 kb in size, with one breakpoint centromeric to the Hoxd cluster disrupting the Lnp gene, and the other breakpoint telomeric to the Hoxd cluster 18 (Fig. 1B). The inversion included Evx2, the Hoxd locus, and Mtx2 and is thus very similar to the centromeric duplication identified in the MDK family.

The pathogenesis of Ul was further clarified by an elegant series of experiments that led to the identification of a HOXD control region located at the 5' (centromeric) side approx. 200 kb away from the 5' end of the HOXD cluster 18 . This so called global control region (GCR, blue oval, fig. 1C and2) positively influences the expression of EVX2 and other 5' HOXD genes expressed in the distal limb and suppresses their expression in the proximal limb. Recently, Tarchini and Duboule specified an additional regulatory element centromeric to the HOXD cluster which controls the posterior restriction of 5' HOXD genes (CenR, red oval, fig. 1C and2). These authors claimed that the GCR preferentially controls expression in the presumptive digit domain. In the case of the Ul inversion the centromeric breakpoint is located between the GCR, the additional centromeric regulator, and the cluster thus separating the cluster from the regulatory influence of the GCR and the additional centromeric regulator. The authors suggested that the disconnection of 5' HOXD genes from the regulator results in a down regulation of 5' HOXD genes in the distal limb (autopod) and an upregulation in the proximal limb. The duplication described here may have a similar effect. Like in the Ul inversion, the duplicated cluster would be located more than 500 kb away from the GCR and the additional centromeric regulator thus escaping its control (fig 2B).

The presence of two breakpoints at the telomeric side of the cluster that result in an MDK-like phenotype suggests an alternative explanation in which the responsible regulator is located 3' of HOXD1. Indeed, Zakany and colleagues propose the presence of an early limb control region (ELCR, green oval, fig. 1C and2) localized telomeric to the cluster which is needed to implement colinear expression of the HOXD cluster 24 .

The exact position of the ELCR is not known. One of the duplications detected in the Thai family is expected to disconnect the 5' located cluster from the 3' located regulator and vice versa. As a consequence HOXD13 is expressed too early and ectopically leading to the MDK phenotype (fig 2B). Thus, it is possible that one or both regulators have to be affected to produce the MDK/Ul phenotype. To verify our results of the Thai family we tested further described, not-related MDK individuals 3,4 . We were not able to identify a clinically relevant copy number change anywhere in the genome of these two additional individuals. However, since the array CGH analysis does not exclude balanced structural rearrangements one can speculate that the MDK phenotype in patients reported by Shears et al. and Kwee et al. 3,4 and the Thai patients reported in this manuscript is caused by chromosomal rearrangements affecting HOXD locus regulation. The identification of further rearrangements in this region will help to clarify

  this. and the inversion result in mesomelic shortening. Regulatory elements are indicated by colored ovals (blue = global control region -GCR; red = centromeric repressor -CenR; green = early limb control region -ELCR).Genomic positions are according to human genome version hg18.

Figure 2 (

 2 Figure2

6

Acknowledgement

We thank the family for participating in this study. This work was supported by grants from the Deutsche Forschungsgemeinschaft to SM and EK. The Thailand Research Fund (TRF) to PK. posterior restriction. As a consequence HOXD13 is expressed too early and ectopically leading to the MDK phenotype. The later HOXD expression in the digit domain controlled by the GCR is unaffected by the genomic duplications.

Conflict of interest

The authors declare no conflict of interest. 16,17 . The translocation t(2;8)

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