Karin Frank-Raue

Lisa A Rybicki

Zoran Erlic

Heiko Schweizer

Aurelia Winter

Ioana Milos

Sergio Pa Toledo

Rodrigo A Toledo

Marcos R Tavares

Maria Alevizaki

Caterina Mian

Heide Siggelkow

Michael Hüfner

Nelson Wohllk

Giuseppe Opocher

Šárka Dvořáková

Bela Bendlova

Małgorzata Czetwertynska

Elŝbieta Skasko

Marta Barontini

Gabriela Sanso

Christian Vorländer

Ana Luiza Maia

Attila Patocs

Thera P Links

Jan Willem De Groot

Michiel N Kerstens

Gerlof D Valk

Konstanze Miehle

Thomas J Musholt

Josefina Biarnes

Svetozar Damjanovic

Mihaela Muresan

Christian Wüster

Martin Fassnacht

Mariola Peczkowska

Christine Fauth

Henriette Golcher

Martin A Walter

Josef Pichl

Friedhelm Raue

Charis Eng

Hartmut Ph Neumann
email: hartmut.neumann@uniklinik-freiburg.de

MD Hartmut P H Neumann

Risk profiles and penetrance estimations in multiple endocrine neoplasia type 2A caused by germline RET mutations located in exon 10

Keywords: Multiple endocrine neoplasia type 2, medullary thyroid carcinoma, pheochromocytoma, genotype-phenotype correlation 2, medullary thyroid carcinoma, pheochromocytoma, genotype-phenotype correlation

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For exon 10, comprehensive molecular and corresponding phenotypic data are scarce. The International RET Exon 10 Consortium, comprising 27 centers from 15 countries, analyzed patients with RET exon 10 mutations for clinical risk profiles.

Presentation, age-dependent penetrance and stage-at-presentation of medullary thyroid carcinoma (MTC), pheochromocytoma and hyperparathyroidism were studied. A total of 340 subjects from 103 families, age 2-86, were registered. There were 21 distinct single nucleotide germline mutations located in codons 609 (45 subjects), 611 (50), 618 (94), and 620 (151). MTC was present in 263 registrants, pheochromocytoma in 54 and hyperparathyroidism in 8 subjects. Of the patients with MTC, 53% were detected when asymptomatic, and amongst those with pheochromocytoma, 54%. Penetrance for MTC was 4% by age 10, 25% by 25 and 80% by 50. Codon-associated penetrance by age 50 ranged from 60% (codon 611) to 86% (620). More advanced stage and increasing risk of metastases correlated with mutation in codon position (609->620) near the juxtamembrane domain. Our data provide rigorous bases for timing of pre-morbid diagnosis and personalized treatment/prophylactic procedure decisions depending on specific RET exon 10 codons affected.

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Introduction

Multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominant multiglandular tumor syndrome, affecting derivatives from neural crest. MEN 2 is caused by germline mutations of the RET proto-oncogene (OMIM164761), encoding a transmembrane receptor tyrosine kinase expressed by the C-cells of the thyroid, adrenal medullary cells, and enteric autonomic ganglia (Elisei, et al., 2007;Eng C, 1996 ;Zbuk and Eng, 2007). When RET mutations were found in the large majority of MEN 2 patients and a RET genotype-clinical phenotype found (Eng C, 1996 ), this was the paradigm for the practice of clinical cancer genetics (Kloos, et al., 2009;Zbuk and Eng, 2007). Accurate molecular diagnosis, pre-morbid predictive testing and geneinformed medical management became possible.

Based on combinations of clinical manifestations, MEN 2 is historically classified into three clinical subtypes, MEN 2A (MIM# 171400), MEN 2B (MIM# 162300), and familial medullary thyroid carcinoma (FMTC, MIM# 155240). The components of MEN 2A are medullary thyroid carcinoma (MTC), pheochromocytoma and parathyroid adenoma and/or hyperplasia (HPT). In MEN 2B, constitutional abnormalities such as marfanoid habitus and neuromas of the tongue and the intestine are present whereas clinical parathyroid disease is absent. FMTC refers to the familial occurrence of MTC without other lesions (Kloos, et al., 2009). MEN 2B is considered more aggressive than MEN 2A or FMTC, because the age-of-onset of the neoplasias is a mean 10 years earlier than that of MEN 2A, and metastases from MTC occur as early as 3 years of age (Kloos, et al., 2009).

The spectrum of germline RET mutations in MEN 2 are encompassed by 64 distinct mutations that lie in 32 codons belonging to 8 exons (Kloos, et al., 2009) . It is clear that the MEN 2B-defining mutations, M918T and A883F, portend an aggressive Deleted: (Kloos, et al., 2009;Zbuk and Eng, 2007 course, with management appropriately aggressive and pre-emptive (Kloos, et al., 2009). It is also obvious and relatively consistent that the exon 11 C634R, the most common mutation seen in MEN 2A, predicts for the development of MTC, pheochromocytoma and HPT, at relatively young ages, and so management is tailored accordingly (Eng C, 1996 ;Frank-Raue, et al., 2006;Kloos, et al., 2009;Neumann, et al., 2002). However, apart from vague generalizations such as non-634 mutations are not as aggressive, perhaps have later ages of onset and decreased penetrance, the breadth of clinical presentations and course, and the less-than-firm data preclude clinicians from altering medical management based on precise singlecodon/specific missense mutation genotype. For instance, studies have shown that mutations at codon 634, the most frequently mutated in MEN 2, result in a receptor tyrosine kinase that is constitutively active by the tendency of mutant RET monomers to undergo dimerization, a situation that simulates ligand binding. Whereas cysteine residues in wild-type RET form intramolecular disulfide bonds, mutation of a cysteine residue leaves an unpaired residue. Unpaired cysteines from two mutant RET monomers can dimerize by formation of a disulfide bond. Taking into account the clinical presentation, we know that the C634R mutation is highly penetrant and associated with the more severe forms of MEN 2A (Eng C, 1996 ;Kloos, et al., 2009). Similarly, while investigating pheochromocytoma presentations in our Registry, we noted that MTC does not always occur before other component neoplasias in those with C634W mutations (Neumann, et al., 2007). Therefore, we believe that single nucleotide genotype-specific clinical risk profiles comprising risk of developing each component neoplasia, the ages-at-onset, penetrance and clinical course might be useful for tailoring more precise clinical care.

In contrast to well-defined risk profiles for carriers of the C634R, C634Y, C634W, A883F, and M918T mutations, mutations located in the codons of RET exon 10 are relatively rare (Mian, et al., 2009;Moers, et al., 1996;Siggelkow, et al., 2001), and their anecdotal penetrance and ages-of-onset have resulted in appropriately cautious and tentative non-consensual clinical guidelines (Brandi, et al., 2001;Kloos, et al., 2009). Here, concerted effort from an international consortium have resulted in RET exon 10 genotyping, documented phenotypes-at-presentation and longitudinal clinical data with the purpose of generating codon-and nucleotide-specific genotypeadjusted neoplasia risk profiles for exon 10 mutations.

Patients and Methods

Patients

The inclusion criterion for registrants was a proven carrier status of a germline mutation in exon 10 of the RET gene. In addition, relatives of index registrants were also registered if an MTC or a pheochromocytoma was diagnosed histologically. The ascertainment of carriers of such mutations was performed in 3 steps. First, RET exon 10 mutation carriers were identified from existing registrants in the European-American Pheochromocytoma Registry (EAPR), which has been previously described (Erlic, et al., 2009;Neumann, et al., 2002;Neumann, et al., 2004;Neumann, et al., 2007;Schiavi, et al., 2005). Second, all clinicians and researchers who had contributed to this registry or to our previous risk estimation study of RET C634W carriers were contacted (Milos, et al., 2008). Finally, all known centers with clinical activities in MEN 2 or one of the component neoplasias (eg, MTC or pheochromocytoma) were asked to contribute any relevant data to this study.

Clinical Data

Recording of demographic and clinical information was performed using a uniform data-format for all registrants. Demographic data included year of birth, gender, ethnic background and country of residence. Clinical data were recorded for thyroid, adrenal and parathyroid glands, and additional features of MEN 2, as follows. For all three components, i.e., MTC, pheochromocytoma and HPT, the age(s)-atdiagnosis were registered using the age at histological diagnosis. Criteria for absence of MTC included normal basal serum calcitonin or histological absence in surgical thyroid specimens. Absence of pheochromocytoma was documented either by normal CT or MRI of the adrenal glands or by normal 24-hour catecholamine excretion. Normal serum calcium and parathyroid hormone levels denoted an absence of hyperparathyroidism.

We differentiated symptomatic patients and subjects who were identified by screening procedures (screening assessment). Screening procedures included genetic screening of relatives after a RET mutation was identified and verified in an index case. This was followed by clinical screening if the mutation was also found in that relative. In addition, relatives may have already had clinical screening following the identification of MEN 2 or one of its principal components in an index case.

For histopathology of the thyroid, we recorded presence or absence of C-cell hyperplasia and of MTC. For lymph node status, we recorded presence or absence of metastases at thyroid surgery. Distant metastases were recorded at thyroid surgery or any time during follow up. classification (DeLellis RA, 2004;Greene LF, 2002;Sobin LH, 2002). T1N0M0 for MTC<2cm in diameter, T2N0M0 for MTC of 2-4cm in diameter, T3 >4cm either limited to the thyroid or with minimal invasion to the sternocleidomastoid muscle or perithyroidal soft tissue, T4a any size with invasion to subcutaneous tissue, larynx, trachea, esophagus or recurrent laryngeal nerve, T4b invasion of prevertebral fascia, carotid artery or mediastinal vessels, N1 for lymph node metastases and M1 for distant metastases. In cases of persistent or clearly elevated calcitonin levels after thyroidectomy including regional lymph node dissection, we classified 6 patients with distant metastases, although radiological evidence was not present.

This study has been approved by the respective institutional review boards for human subjects' protection in accordance with the ethical standards of each country and center.

Statistical Analysis

Categorical variables were summarized as frequency counts and percentages.

Continuous variables were summarized as the median and range. MTC stage, cure/no-cure, and presence/absence of metastasis were compared among codons. variables were included in a multivariate logistic regression model whether or not they were statistically significant. Age-dependent penetrance estimates of MTC, pheochromocytoma and HPT were calculated using the Kaplan-Meier method.

Penetrance estimates were compared among codons using the log-rank test and with Cox proportional hazards analysis. The log-rank analysis assesses penetrance differences among codons, while the Cox analysis assesses trends across codons. Some patients had incomplete data regarding the different manifestations and were excluded from penetrance estimates. Thus, the penetrance data for MTC are based on 340 subjects, pheochromocytoma 319 subjects, and HPT 299 subjects. All analyses were done using SAS® software (SAS® Institute, Inc., Cary, NC, USA). All statistical tests were two-sided, and P<0.05 was used to indicate statistical significance.

Results

Overall, 340 individuals, 199 females and 141 males, had germline mutations in exon 10 of the RET gene and their clinical phenotypes and penetrance were analyzed for purposes of this study. The mutations affected only cysteine codons 609, 611, 618 and 620 (Table 1). The 340 subjects, 255 European and 85 South American (comprising 280 of Caucasian, 20 of Hispanic, 3 of Italian-German-Hispanic, 37 of Indian-Caucasian and 1 of Gypsy origin), belonged to 103 different families and hailed from 14 different countries (Table 1). Each of the centers contributed from 1 to 43 cases. The center specific distributions of cases amongst the codons is shown in Table 2 Pheochromocytoma was observed in individuals with mutations of all four codons. However, pheochromocytoma was not observed in carriers of 7 of the 22 distinct mutations. The frequencies of pheochromocytoma varied, occurring in 26% of those patients with codon 609 mutations, 10% codon 611, 23% codon 618 and 13% codon 620 (Table 1). All pheochromocytomas were adrenal in location. Bilateral pheochromocytoma was present in 15 of 54 (28%) affected subjects. None had malignant pheochromocytoma. Symptomatic pheochromocytoma occurred in 29 (54%) of the 54 patients; 24 of these 29 also had MTC. Among these 24, pheochromocytoma was diagnosed at least one year prior to MTC in 6 patients (25%), within 1 year of MTC in 8 (33%), and at least one year after MTC in the remaining 10 (42%).

HPT was present in 8 of 299 subjects (2.7%). Median age at diagnosis of HPT was 46 years (range 28-82). Of the 8 with HPT, 7 were ascertained by screening at an asymptomatic stage. Although sample size is small, HPT was found in 4 out of 22 distinct mutations residing in all four exon 10 codons.

Regarding codon-specific neoplasias seen in our MEN 2 series, MTC and pheochromocytoma were present in carriers of germline mutations of each of the four codons. Notably, codon 609 mutation carriers as a group have similar frequencies of MTC and pheochromocytoma as the groups carrying mutations in codons 611, 618 and 620 (p=0.2, Fisher 2-tailed exact test). However, if we analyzed the frequencies of MTC and pheochromocytoma for each distinct mutation, FMTC was found in patients carrying 6 specific missense mutations distributed across all 4 codons (Table 1), although sample sizes per nucleotide change are small. In contrast, none of the 340 subjects had signs of MEN 2B.

Age-related Penetrance for RET Exon 10 Mutations

Age-related penetrance for each MEN 2 component neoplasia was estimated for all the subjects as well as for each of the 4 cysteine codons. Overall for all exon 10 mutations, considering both symptomatic and screening ascertained cases, 50% penetrance was achieved by the age of 36 years for MTC, by 68 years for pheochromocytoma, and by 82 years for HPT (Fig. 1).

Age-related penetrance of MTC for mutations in codons 609, 611, 618, and 620 is shown in detail in table 3. Fifty % penetrance was achieved by the ages of 40, 44, 35, and 34 years for mutations in codons 609, 611, 618 and 620, respectively.

Penetrance curves were significantly different among the four codons (p=0.018, logrank test; Fig. 2), and there was also a significant trend across the four codons (p=0.010, Cox analysis) whereas within each of the affected codons, penetrance for MTC was not different for the given mutations (data not shown). The latter may be due to small sample sizes due to subset analysis. We also compared age-related penetrance between symptomatic and screening ascertainment for MTC and found they were not different (p=0.90). The ages at 50% penetrance for MTC was 36 years among those symptomatically ascertained and 35 years among those ascertained by screening. Similarly, the agerelated penetrance at age 50 years was 83% and 76%, respectively.

For 319 subjects in whom pheochromocytoma was assessed, age-related penetrance at 50 years was 23% (Fig. 1 and Table 3). Penetrance curves were nearly different among codons (p=0.08, log-rank test; Fig. 3), but there was no trend in pheochromocytoma penetrance across the four codons (p=0.99, Cox analysis).

Pheochromocytoma ascertainment was only known in 156 patients; however, penetrance was significantly different between pheochromocytoma ascertained from symptomatic assessment and by screening (p<0.001; Fig. 4). Fifty % penetrance was seen by 40 years for 29 symptomatic patients, and by 65 years for 127 screened patients, while penetrance by age 50 was 76% and 23%, respectively.

MTC Tumor Stage

TNM classification was recorded for 300 subjects. Age at diagnosis of any MTC is summarized by codon in table 1. The youngest age at manifestation of MTC in these patient groups is 4, 14, 5, and 6 years for codons 609, 611, 618, and 620, respectively. The youngest age having lymph node metastasis was 21, 29, 9, and 16 years for the respective codons (Table 3). Penetrance for localized MTC was relatively high (67.3%) by the age of 60 for all exon 10 mutations with the highest penetrance for regional nodal involvement and distant metastases for codon 620 mutations (Table 3). Penetrance differed significantly among codons (p=0.005, logrank test), and there was also a trend across the four codons (p=0.003, Cox analysis). In addition penetrance for the very early stage of C cell hyperplasia (CCH), as well for distant metastases (M1) are shown in table 3. CCH penetrance was not different among codons (p=0.16, log-rank test), nor was there a trend across codons (p=0.10, Cox analysis). M1 penetrance was not quite different among codons (p=0.12, log-rank test), but there was a trend across codons (p=0.020, Cox analysis).

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Comparing MTC stages, i.e. metastasized or not metastasized and cured or not cured among codons using the Chi-square test showed a significant difference among codons (Table 4). When ordered by codons, 609, 611, 618 and 620, higher stages (p<0.001), increased the likelihood of metastasis (p=0.002), and decreased the chance of cure (p<0.001). Death caused by MTC was documented in 0, 1, 6, and 11 patients in the codons 609, 611, 618, and 620, respectively. Gender, age, and mutated codon were examined as possible risk factors for MTC. In univariable Chi-square analysis, female gender (p=0.008) and older age (p<0.001) were associated with higher risk of MTC. Specific codon mutated was not quite significant (p=0.06); there appeared to be no trend across codons, but rather codon 618 mutation had the highest risk of MTC (76% MTC for codon 609, 72% for 611, 87% for 618, and 74% for 620). In multivariable analysis, gender becomes nonsignificant, age remains significant (p<0.001), and codon is not quite significant, but again, codon 618 has the highest risk of MTC (p=0.07).

Discussion

Because the prevalence of RET exon 10 mutations, even all together, is much lower than that of the exon 11 codon 634 mutations, information on the natural history and age-related penetrance for component neoplasias remained scarce and was provided mostly by reports from single centers or on single large families (Calva, et al., 2009;Mian, et al., 2009;Moers, et al., 1996;Siggelkow, et al., 2001). The Deleted: Lisas suggestion for the sentence highlighted in blue: Penetrance differed significantly among codons (p=0.005, log-rank test), and there was also a trend across the four codons (p=0.003, Cox analysis). In addition penetrance for the very early stage of C cell hyperplasia (CCH), as well for distant metastases (M1) are shown in table 3. CCH penetrance was not different among codons (p=0.16, log-rank test), nor was there a trend across codons (p=0.10, Cox analysis). M1 penetrance was not quite different among codons (p=0.12, log-rank test), but there was a trend across codons (p=0.020, Cox analysis). ¶ American Thyroid Association (ATA) has, in particular, addressed this issue as a point of concern. Guidelines for diagnosis and management of MTC and other components of MEN 2 have reached only a weak consensus level for these management guidelines for carriers of mutations in exon 10 (Kloos, et al., 2009). In this present study, we summarize and analyze comprehensive data for subjects carrying such RET exon 10 mutations based on a multicenter, multiethnic and multinational series of 340 individuals who all have been registered using identical criteria. Thus, we are able to obtain molecular-based neoplasia risk profiles and codon-specific age-related penetrance on which to base genetic counseling and medical decision-making.

Our current series demonstrates that 15 distinct mutations involving the 4 codons of RET exon 10 can all be associated with MEN 2A. In contrast, the FMTC phenotype was seen to be associated with 6 distinct mutations, also involving all the 4 codons (Table 1). The studied number of carriers with these 7 mutations ranges from 2 to 26 (mean 11) per mutation. Therefore, the lack of pheochromocytoma in these subjects may be due to sample size and/or penetrance.

Missense mutations nearer the N-terminus are associated more with HSCR based on a clear mechanism (Santoro, et al., 1999). The first comprehensive genotype-phenotype study on MEN 2 suggested that as high as 1/3 of all carriers of mutations in codons 618 and 620 had HSCR (Eng C, 1996 ). However, the sample sizes for exon 10 mutations in that series was relatively small. In even smaller series and single family reports, the prevalence of HSCR in codon 620 mutation carriers approached 50% (Butter, et al., 2007). In the present series, HSCR was only present in 20/267 informative carriers (7.5%), 2/37 (5.4%) in those with mutations in codon 609, 0/44 in codon 611, 1/56 (1.8%) in codon 618, and 17/130 (13.1%) in codon 620 (data not shown). This is a lower prevalence than initially predicted, however, it is tempting to speculate if other intra-RET modifying variants, whether in cis or trans, would modulate the HSCR phenotype in such individuals (Borrego, et al., 1998;Borrego, et al., 2000;Borrego, et al., 1999;Borrego, et al., 2003;Fernandez, et al., 2003).

Even before RET was identified, the classic penetrance of MTC in MEN 2A is cited as 70% by age 70 by symptomatic presentation and 100% by age 70 by stimulated calcitonin screening (Ponder, et al., 1988a;Ponder, et al., 1988b). Not surprisingly, age-related penetrance for codon 634 mutations approach >85% by age 80 for MTC and >50% by age 80 for pheochromocytoma (Iihara, et al., 1997;Kloos, et al., 2009;Milos, et al., 2008;Modigliani, et al., 1998). Mutations in exon 10 were believed to have much lower age-related penetrances. While our series showed that penetrance for MTC was 50% at age 36 for exon 10 mutations which is lower than the 50% by age 28 for the C634W mutation (Milos, et al., 2008), both exon 10 and 11 mutations reach >80% penetrance by age 50 and almost 100% by age 70.

Interestingly, the penetrance for distant metastases from MTC was 9% for p.C634W compared to 36% for codon 620 mutations (Table 3). In contrast, the age-related penetrance of exon 10 mutations for pheochromocytomas was systematically lower than that for codon 634 mutations, such as for p.C634W (33% vs 70% by age 60, Table 3). Notably, there is a significant difference in pheochromocytoma penetrance between those that were symptomatically ascertained compared to those ascertained by screening. This underscores the great importance of clinical screening for pheochromocytoma even in exon 10 mutation carriers, beginning in the late teens or early 20's. Our data support the ATA guidelines recommending initiation of screening for pheochromocytoma at the age of 20 in all mutations except those in codons 918 and 634 respectively; for these mutations, screening for pheochromocytoma is recommended beginning by the age of 8 years. Although sample size was small, the penetrance for HPT was 34% by age 60 for p.C634W compared to 3.9% by age 60 for exon 10 mutations (Table 3).

The results of this multicenter study of 340 patients carrying exon 10 mutations show differences in aggressiveness of MTC depending on the mutated codon. This results in earlier age at manifestation, more advanced tumor stage at manifestation and decreased chance of cure when ordered by codons (620, 618, 611, and 609). In this patient group, age is an independent risk factor for the manifestation of MTC. Position of mutation was associated with metastatic disease, with the lowest likelihood amongst those with codon 609 mutations, and the highest, codon 620 mutations. This is important for the decisions regarding timing of premorbid diagnoses and treatment/prophylactic procedure in RET gene carriers, as age is an independent predictor of aggressive disease at thyroidectomy: MTC is more aggressive and might develop earlier in codons 618 and 620. This supports the clinical impression that patients with RET mutations in codons 609 and 611 are older at presentation (Calva, et al., 2009;Hansen, et al., 2000;Mian, et al., 2009;Siggelkow, et al., 2001), and have more indolent tumors than patients with RET mutations at 618 and 620 (Machens, et al., 2009), but there are notable exceptions as shown in our systematic study. It is important to point out that also a patient with codons 609 mutation can present with MTC by age 4.

Multivariate analysis of our data reveals that age at presentation is associated with higher likelihood of metastases from MTC, a finding that is not surprising. What is also not surprising is that those who were ascertained symptomatically present at more advanced stages compared to those ascertained by screening, where a full 1/3 presents with normal histology or CCH only. Taken together, our observations strongly support clinical surveillance and/or timely prophylactic thyroidectomy even in exon 10 mutation carriers, which is certainly notated in the ATA MTC Practice Guidelines. However, the age to begin these remain unclear as the impression up until recently is exon 10 mutations do not normally confer early ages of onset of MTC.

In our current series, however, the youngest age at diagnosis for MTC was 4 years (in a codon 609 mutation carrier) and for MTC with regional nodal metastases 9 years (in a codon 618 mutation carrier), respectively. Not surprisingly, therefore, those with codons 618 and 620 mutations had the highest prevalence of metastatic MTC, even higher than those with p.C634W. The youngest age for distant metastases amongst exon 10 mutation positive individuals was aged 13 years. The more typical age of MTC development for the exon 10 genotype reveals significant differences in aggressiveness of MTC manifestation e.g. higher penetrance of lymph node metastases and distant metastases in codons 618/620 in comparison to exon 609/611. The suggestion of the ATA guidelines to consider prophylactic surgery in patients carrying exon 10 codon 618 and 620 mutations at age of 5 years seems to be supported by our data. In patients with codon 609 and 611 prophylactic thyroidectomy possibly might be postponed under careful yearly clinical and laboratory examinations.

In summary, we have presented a large series of uniformly collected and registered cases with germline exon 10 mutations in RET, and have analyzed neoplastic risk profiles and age-related penetrance for each component neoplasia, which will help evidence-based genetic counseling and medical management.

  type 2 is characterized by germline mutations in RET.

For

  MTC tumor stages, we used the TNM classification of 2002 and the AJCC

  Comparisons were made with the Chi-square test which determines, if variables differ between at least two of the four codons; and with the Cochran-Mantel-Haenszel mean score test (TNM stage) or Cochran-Armitage trend test (cure, metastasis). These trend tests account for the ordering of codons by distance from the transmembrane domain and assess trends in stage, cure or metastasis. Risk factors for MTC were assessed via Chi-square tests and logistic regression analysis. Three candidate MTC risk factors were assessed: gender, age, and codon. All three

Figure 1 .

 1 Figure Legends

Figure 2 .

 2 Figure 2. Penetrance of medullary thyroid carcinoma (MTC) of 340 carriers adjusted for germline mutations of the codons 609 (n=45 carriers), 611 (n=50 carriers), 618 (n=94 carriers), and 620 (n=151 carriers).

Figure 3 .

 3 Figure 3. Penetrance of pheochromocytoma (Pheo) of 319 carriers adjusted for germline mutations of the codons 609 (n=43 carriers), 611 (n=50 carriers) 618 (n=84 carriers), and 620 (n=142 carriers).

Figure 4 .

 4 Figure 4. Penetrance of pheochromocytoma (Pheo) adjusted for detection by symtomatic pheochromocytoma (Symptomatic) and by screening-detected pheochromocytoma (Screening).

  .Codon 609 mutations were found in 45 subjects from 13 families. Codon 611 mutations were noted in 50 subjects from 12 families. Codon 618 mutations were found in 94 subjects from 37 families. Codon 620 mutations were identified in 151 subjects from 41 families (Table1).

	Phenotypic Spectrum of RET Exon 10 Mutations
	MTC was the most common neoplasia, with 263 affected (77%). In the
	remaining 23%, diagnosis of MTC was excluded by objective means. The median
	age at diagnosis of MTC was 35 (range 4-86). Of note, symptomatic ascertainment of
	MTC occurred in 47% of the 263 patients whereas in 53%, their MTC was detected F by screening. o Pheochromocytoma was documented in 54 of 319 subjects (17%). The r median age at diagnosis of pheochromocytoma was 42 years (17-80).
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Table 1

 1 Demographic and molecular data of the study population

								Human Mutation					Page 34 of 38
	Codon	Nucleotide	Protein	Families n	Carriers n	Gender f/m	Age at MTC diagnosis: median (range)	MTC n (%)	Pheo n (%)	HPT n (%)	HSCR n (%)	Phenotype	Age at N1M0 n (range)	Age at M1 n (range)
		c.1825T>C	C609R	2	7	7/0	42 (29-43)	4/7 (57)	2/7 (29)	0	0	MEN2A	1 (40)	0
		c.1825T>G	C609G	3	6	1/5	48 (4-54)	5/6 (83)	4/5 (80)	0	0	MEN2A	2 (33-48)	0
		c.1826G>A	C609Y	4	8	7/1	33 (21-49)	7/8 (88)	0	0	2/8 (25)	FMTC,HSRC	2 (21-48)	0
	609
		c.1826G>C	C609S	2	18	6/12	37 (15-86)	14/18 (78)	4/17 (24)	2/17 (12)	0	MEN2A	1 (38)	1 (62)
		c.1826G>T	C609F	2	6	5/1	45 (36-63)	4/6 (67)	1/6 (17)	0	0	MEN2A	1 (54)	0
		All		13	45	26/19	37 (4-86)	34/45 (76)	11/43 (26)	2/44 (5)	2/37 (5)	MEN2A,HSRC	7 (21-54)	1 (62)
		c.1832G>A	C611Y	7	15	11/4	35 (15-65)	9/15 (60)	4/15 (27)	1/15 (7)	0	MEN2A	4 (29-64)	1 (65)
		c.1833C>G C611W	2	5	4/1	37 (14-66)	5/5 (100)	1/5 (20)	0	0	MEN2A	1 (49)	1 (37)
	611	c.1832G>T	C611F	2	26	12/14	44 (25-69)	19/26 (73)	0	0	0	FMTC	1 (44)	5 (27-69)
		c.1832G>T
			C611F	1	4	3/1	53 (36-64)	3/4 (75)	0	0	0	FMTC	0	0
		c.1833C>T
		All		12	50	30/20	42 (14-69)	36/50 (72)	5/50 (10)	1/44 (2)	0	MEN2A	6 (29-64)	7 (27-69)
	618	c.1852T>A	C618S	4	13	8/5	35 (18-59)	10/13 (77)	3/12 (25)	0	0	MEN2A	2 (42-59)	2 (31-39)
		c.1852T>C	C618R	17	28	22/6	35 (10-65)	28/28 (100)	11/28 (39)	0	1/12 (8)	MEN2A	11 (21-45)	7 (13-65)
		c.1852T>G	C618G	3	6	4/2	30 (21-57)	5/6 (83)	0	0	0	FMTC	0	1 (57)
		c.1853G>C	C618S	7	14	4/10	27 (9-63)	12/14 (86)	2/13 (15)	0	0	MEN2A	4 (9-50)	0
							John Wiley & Sons, Inc.

Table 3 Penetrance data for RET codon 10 mutations compared to the C634W mutation of codon 11 adjusted for C cell hyperplasia (CCH), MTC stage T1- 4N0M0, stage T1-4N1M0, and stage T1-4N0or1M1, and for pheochromocytoma (pheo) and hyperparathyroidism (HPT)

 3

	Table 3 (part 1)
	CCH

Table 2 Comparison of MTC-tumor-stage and outcome in the 4 mutated codons in exon 10

 2

	609	611	618	620	p-value
	n (%)	n (%)	n (%)	n (%)

John Wiley & Sons, Inc.Human Mutation

This study is funded in part by grants from the Deutsche Krebshilfe (70-3313-Ne 1 to HPHN), the Deutsche Forschungsgemeinschaft (NE 571/5-3 to HPHN), the European Union (LSHC-CT-2005-518200 to HPHN). RAT was supported by a Sao Paulo State Research Foundation-FAPESP grant (2009/15386-6), and SPAT by a National Research Foundation-CNPq grant (310614/09) CE was a recipient of the Doris Duke Distinguished Clinical Scientist Award, holds the Sondra J. and Stephen R. Hardis Endowed Chair of Cancer Genomic Medicine at the Cleveland Clinic, and is the American Cancer Society Clinical Research Professor, generously funded in part by the F. M. Kirby Foundation.

ALM is a recipient of a National Research Foundation-CNPq grant (305629/07).


Fransiska ; Malfait
email: fransiska.malfait@ugent.be

Delfien Syx

Philip Vlummens

Sofie Symoens

Sheela Nampoothiri

Trinh Hermanns-Lê

Lut Van Laer

Anne Depaepe

Lut Van Laer

Anne De Paepe

Ehlers-Danlos syndrome (former EDS type VIB) and adducted thumb clubfoot syndrome (ATCS) represent a single clinical entity caused by mutations in the dermatan-4-sulfotransferase 1 encoding CHST14 gene

Keywords: Ehlers-Danlos syndrome, collagen, CHST14, dermatan-4sulfotransferase 1, adducted thumb clubfoot syndrome Ehlers-Danlos syndrome type VI, collagen, CHST14, dermatan-4-sulfotransferase 1, adducted thumb clubfoot syndrome gastro-intestinal and genitourinary manifestations, c.145delG, p.V49X c.981_1000dup, pGlu334GlyfsX107

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

The Ehlers-Danlos syndrome (EDS) comprises a heterogeneous group of connective tissue diseases of which the major clinical features are skin hyperextensibility, joint hypermobility and generalized connective tissue fragility. [START_REF] Steinmann | The Ehlers-Danlos Syndrome[END_REF] The current classification recognizes six subtypes which differ in clinical symptoms, inheritance pattern and the nature of the underlying biochemical and molecular defect(s). In several of these subtypes mutations have been identified in genes encoding the polypeptide (α)-chains of fibrillar collagen type I (arthrochalasia type), collagen type III (vascular type) and collagen type V (classic type), or in genes encoding enzymes involved in the posttranslational modification of type I collagen (kyphoscoliotic and dermatosparaxis type). [START_REF] Beighton | Ehlers-Danlos syndromes: revised nosology, Villefranche, 1997. Ehlers-Danlos National Foundation (USA) and Ehlers-Danlos Support Group (UK)[END_REF]. Recently, several new EDSvariants have been characterized, which include the progeroid form of EDS (β4GALT7, OMIM 604327 ) [START_REF] Quentin | A genetic defect in the biosynthesis of dermatan sulfate proteoglycan: galactosyltransferase I deficiency in fibroblasts from a patient with a progeroid syndrome[END_REF]; a Tenascin-X deficient form of EDS (TNXB, OMIM 600985) [START_REF] Schalkwijk | A recessive form of the Ehlers-Danlos syndrome caused by tenascin-X deficiency[END_REF]; a cardiac-valvular EDS-form (COL1A2, OMIM 120160) [START_REF] Schwarze | Rare autosomal recessive cardiac valvular form of Ehlers-Danlos syndrome results from mutations in the COL1A2 gene that activate the nonsense-mediated RNA decay pathway[END_REF][START_REF] Malfait | Total absence of the alpha2(I) chain of collagen type I causes a rare form of Ehlers-Danlos syndrome with hypermobility and propensity to cardiac valvular problems[END_REF]; and an EDS-like spondylocheirodysplastic form (SLC39A13 OMIM 608735). [START_REF] Giunta | Spondylocheiro dysplastic form of the Ehlers-Formatted: Line spacing: Double Field Code Changed Formatted: English (U.K.) Formatted: German[END_REF] EDS kyphoscoliotic type or EDS type VI is an autosomal recessive (AR) disorder which is characterized by early-onset progressive kyphoscoliosis, severe neonatal muscle hypotonia with delayed gross motor development, generalized joint hyperlaxity, marfanoid habitus, osteopenia, and a fragile, hyperextensible and bruisable skin with widened atrophic scars. In some patients, scleral fragility with risk for rupture of the globe, and life-threatening rupture of medium-sized arteries have occasionally been reported. (Yeowell and Steinmann, 2000) One form of EDS kyphoscoliotic type, EDS type VIA, is caused by deficient activity of the enzyme procollagenlysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1 or lysyl hydroxylase-1 (LH-1)) (Pinnell et al., Deleted: [START_REF] Steinmann | The Ehlers-Danlos Syndrome[END_REF] Deleted: s Deleted: [START_REF] Beighton | Ehlers-Danlos syndromes: revised nosology, Villefranche, 1997. Ehlers-Danlos National Foundation (USA) and Ehlers-Danlos Support Group (UK)[END_REF] Deleted: [START_REF] Quentin | A genetic defect in the biosynthesis of dermatan sulfate proteoglycan: galactosyltransferase I deficiency in fibroblasts from a patient with a progeroid syndrome[END_REF] Deleted: [START_REF] Schalkwijk | A recessive form of the Ehlers-Danlos syndrome caused by tenascin-X deficiency[END_REF] Deleted: [START_REF] Malfait | Total absence of the alpha2(I) chain of collagen type I causes a rare form of Ehlers-Danlos syndrome with hypermobility and propensity to cardiac valvular problems[END_REF][START_REF] Schwarze | Rare autosomal recessive cardiac valvular form of Ehlers-Danlos syndrome results from mutations in the COL1A2 gene that activate the nonsense-mediated RNA decay pathway[END_REF] Deleted: [START_REF] Giunta | Spondylocheiro dysplastic form of the Ehlers-Formatted: Line spacing: Double Field Code Changed Formatted: English (U.K.) Formatted: German[END_REF] F o r P e e r R e v i e w 4 1972). This enzyme hydroxylates lysyl residues in Xaa-Lys-Gly-triplets of the helical region of collagens and collagen-like sequences of non-collagenous proteins [START_REF] Kivirikko | Collagen hydroxylases and the protein disulfide isomerase subunit of prolyl 4-hydroxylases[END_REF]. The resulting hydroxylysyl residues are essential for the formation of stable intermolecular crosslinks that provide tensile strength and mechanical stability to the collagen fibrils, and they serve as attachment sites for carbohydrate units. These carbohydrate units have been shown to influence the lateral packing of collagen molecules into fibrils. The diagnosis of EDS type VIA can be confirmed by the demonstration of an increased ratio of lysylpyridinoline (LP) to hydroxylysylpyridinoline (HP) crosslinks in the urine, [START_REF] Steinmann | Urinary pyridinoline cross-links in Ehlers-Danlos syndrome type VI[END_REF], a reduction of > 75% of LH-1 enzyme activity in skin (Yeowell and Walker, 2000), and mutation analysis of the PLOD1 gene (OMIM 153454).

A subset of EDS type VI patients have been reported with normal LH-1 activity and normal urinary LP/HP ratios. They are usually referred to as "EDS type VIB" and the underlying molecular defect is hitherto unknown [START_REF] Steinmann | Ehlers-Danlos syndrome in two siblings with deficient lysyl hydroxylase activity in cultured skin fibroblasts but only mild hydroxylysine deficit in skin[END_REF][START_REF] Ogur | Clinical, ultrastructural and biochemical studies in two sibs with Ehlers-Danlos syndrome type VI-B-like features[END_REF][START_REF] Steinmann | The Ehlers-Danlos Syndrome[END_REF]Walker et al., 2004;[START_REF] Kosho | Ehlers-Danlos syndrome type VIB with characteristic facies, decreased curvatures of the spinal column, and joint contractures in two unrelated girls[END_REF][START_REF] Kosho | A new Ehlers-Danlos syndrome with craniofacial characteristics, multiple congenital contractures, progressive joint and skin laxity, and multisystem fragility-related manifestations[END_REF]. A small proportion of these present with predominant corneal fragility and fit into the Brittle Cornea Syndrome (BCS), for which ZNF469 was recently identified as the causal gene. [START_REF] Al-Hussain | Brittle cornea syndrome and its delineation from the kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VI): report on 23 patients and review of the literature[END_REF][START_REF] Abu | Deleterious mutations in the Zinc-Finger 469 gene cause brittle cornea syndrome[END_REF] Here we report the clinical, biochemical and ultrastructural features of three patients with EDS type VIB from two consanguineous families and provide evidence that this phenotype is caused by mutations in CHST14 (OMIM 608429), encoding dermatan-4-sulfotransferase 1. Deleted: (Pinnell, et al., 1972) Deleted: [START_REF] Steinmann | Urinary pyridinoline cross-links in Ehlers-Danlos syndrome type VI[END_REF] Deleted: (Kosho, et al., 2010;[START_REF] Kosho | Ehlers-Danlos syndrome type VIB with characteristic facies, decreased curvatures of the spinal column, and joint contractures in two unrelated girls[END_REF][START_REF] Ogur | Clinical, ultrastructural and biochemical studies in two sibs with Ehlers-Danlos syndrome type VI-B-like features[END_REF][START_REF] Steinmann | Ehlers-Danlos syndrome in two siblings with deficient lysyl hydroxylase activity in cultured skin fibroblasts but only mild hydroxylysine deficit in skin[END_REF][START_REF] Steinmann | The Ehlers-Danlos Syndrome[END_REF]Walker, et al., 2004) Deleted: [START_REF] Abu | Deleterious mutations in the Zinc-Finger 469 gene cause brittle cornea syndrome[END_REF][START_REF] Al-Hussain | Brittle cornea syndrome and its delineation from the kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VI): report on 23 patients and review of the literature[END_REF])

Material and Methods

Subjects

F o r P e e r R e v i e w 5
The patients and parents participated in this study following informed consent. This study has been approved by the Ethics Committee of the Ghent University Hospital, Ghent, Belgium. In all patients, a skin biopsy was taken for ultrastructural and/or biochemical studies. Blood samples were collected from the patients, their parents, and from unaffected siblings.

Biochemical analysis

A dermal fibroblast culture of patient 1 and 2 was established from a skin biopsy, and grown under standard conditions. At confluence the cells were labeled with 14 C proline as described previously [START_REF] Nuytinck | Substitution of glycine-661 by serine in the alpha1(I) and alpha2(I) chains of type I collagen results in different clinical and biochemical phenotypes[END_REF] and collagen proteins were separated on SDS-polyacrylamide gels. The gels were processed for fluorography, dried and exposed to an X-ray film.

Urinary lysylpyridinoline and hydroxylysylpyridinoline excretion was evaluated with HPLC in all patients as described previously. [START_REF] Steinmann | Urinary pyridinoline cross-links in Ehlers-Danlos syndrome type VI[END_REF]

Ultrastructural analyses

For ultrastructural studies, a skin biopsy from the upper arms of patient 2 was fixed with 4% glutaraldehyde in phosphate buffer at pH 4.7 and prepared for transmission microscopy.

Homozygosity mapping

Genomic DNA was isolated from venous blood using the Puregene® DNA purification kit (Gentra Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Genotyping was performed for individuals II.1-3 of family 1 using the Mapping 250K Nsp array from Affymetrix containing 262264 SNPs (analysis performed by DNA Vision, Gosselies,

Belgium). Genomic regions harbouring 25 consecutive homozygous SNPs (approximately 250

Field Code Changed Field Code Changed Deleted: [START_REF] Nuytinck | Substitution of glycine-661 by serine in the alpha1(I) and alpha2(I) chains of type I collagen results in different clinical and biochemical phenotypes[END_REF] Deleted: [START_REF] Steinmann | Urinary pyridinoline cross-links in Ehlers-Danlos syndrome type VI[END_REF] F o r P e e r R e v i e w 6 kb) or more were marked. To overcome SNP miscalls, regions spanning maximum 50 kb between two marked regions were considered homozygous. Eventually, regions that contained at least 100 consecutive homozygous SNPs were assigned as a candidate region if they were homozygous for the same allele in both affected patients and had a different genotype in the nonaffected individual.

Mutation screening

PCR amplification was performed with 50ng genomic DNA. Reaction conditions, oligonucleotide primer pairs and thermal cycling conditions are listed in Suppl. Table S1. PCR products were bidirectionally sequenced using the BigDye® Terminator Cycle Sequencing kit protocol (Applied Biosystems, Foster City, CA, USA), followed by separation on the ABI 3730XL Genetic Analyzer (Applied Biosystems).

Results

Clinical findings

The clinical findings are summarized in Suppl. Table S2 and Patient 1 was born by normal vaginal delivery at 42 weeks of gestation as the second child to first-cousin Turkish parents. Birth weight was 3100g (-0.67 SD) and birth length 51cm (mean).

Occipitofrontal circumference (OFC) was not recorded. She presented a large fontanel, flexionadduction contractures of the thumb, which disappeared during the first year of life, camptodactyly of the 3 rd , 4 th and 5 th finger and clubfeet, which were surgically corrected at age 2,5 yrs. She had a slender build with low muscle and fat mass leading to severe decubitus worsened rapidly at time of puberty and required surgery at age of 14 yrs. Her tissues were noted to be very thin and fragile upon surgical procedures. Skin fragility, delayed wound healing, easy bruising and bleeding from the gums were prominent. An umbilical hernia was corrected at age 5 yrs. Hiatal hernia with gastro-esophageal reflux and frequent bouts of abdominal cramping pain were reported. Abdominal ultrasonography revealed nephrolithiasis at a very young age. Earlyonset high myopia and glaucoma required several surgical treatments, but evolved to phtysis in the right eye and were complicated by retinal detachment in the left eye.

Examination at age 22 yrs revealed a height of 155cm (-2.0 SD) with an armspan of 149 cm and OFC of 55 cm (mean). She presented a slender build with low muscle mass and marked muscular hypotonia, a corrected kyphoscoliosis and a narrow, flattened anterior thorax with no breast development. She had no light perception, and presented mild bilateral hearing loss.

Craniofacial abnormalities included brachycephaly, asymmetric face with deep-set eyes, downslanting palpebral fissures, low-set and posteriorly rotated ears, malar hypoplasia, a short nose with hypoplastic columella, small mouth with thin upper lip, dental crowding, high and narrow palate and a protruding jaw with pointed chin. Her skin was very thin, transparent and hyperextensible, showed several cigarette paperscars and felt doughy and soft upon palpation with a very puffy aspect on lower limbs and feet. She had tapering fingers, severe wrinkling of the palms, flexion contractures of fingers (Fig 1D) and toes, and dislocation of the radio-ulnar joints. Joint hyperlaxity was limited to the metacarpophalangeal joints. She presented talipes valgus and planus. Echocardiography at age 21 yrs was normal, with an aortic root of 17 mm. Patient 2, the younger sister of patient 1, was born at 42 weeks of gestation after a normal pregnancy and vaginal delivery. At birth, her fontanel was large and she had clubfeet which were corrected at 12 months. Muscular hypotonia caused delayed gross motor development and inability to walk independently before age 2 yrs. During childhood, she suffered a spontaneous rupture of the abdominal muscles and diastasis recti, repetitive painful dislocations of the patellae, requiring surgical fixation, and repeated dislocations of temporomandibular, shoulder and ankle joints. At the age of 14 yrs she developed a rapidly progressive lumbar scoliosis. Skin fragility, delayed wound healing, easy bruising and formation of widened atrophic scars were comparable to her sister's. She suffered from early onset nephrolithiasis and abdominal ultrasonography disclosed hydronephrosis due to renal ptosis and ureteral stenosis for which an ureteral stent was placed. The laparoscopic procedure was complicated by severe hemorrhage due to excessive tissue fragility. Early-onset high myopia and glaucoma were complicated by retinal detachment of the left eye. Clinical examination at the age of 14 yrs showed a similar, though milder phenotype as her older sister, with low muscle mass, muscular hypotonia and a narrow, flat thorax. She was 168 cm tall (0.68 SD) with an armspan of 157 cm and an OFC of 58 cm (> 2.0 SD). Facial appearance was characterized by brachycephaly, malar hypoplasia, bushy eyebrows and synophrys, downslanting palpebral fissures, blue sclerae, microcornea, thin upperlip and a high, narrow palate (Fig1A). Her skin had the same doughy, hyperextensible and thin aspect as her sister's, with atrophic cigarette paperscars on both knees (Fig 1

Morphological and ultrastructural studies

The majority of the collagen bundles were small-sized. Some bundles were composed of variable diameter collagen fibrils separated by irregular interfibrillar spaces. Flower-like collagen fibrils were present. Granulo-filamentous material was found throughout the dermis, within the

Biochemical studies

SDS-PAGE analysis of procollagens and pepsin-digested collagens from the medium and the cell layer fraction showed a normal electrophoretic pattern for type I, III and V (pro)collagen proteins (data not shown).

In all three patients, normal LP/HP ratios were observed, excluding the diagnosis of EDS type VIA.

Molecular studies

After excluding the presence of disease-causing mutations in the PLOD1 gene by means of direct sequencing at gDNA level, homozygosity mapping for individuals II.1-3 from family 1 identified four candidate regions which were homozygous among the two affected patients, but not in the non-affected sib and which contained in total 217 known or predicted genes, excluding pseudogenes (NCBI MapViewer, Build 36.3) (detailed description of the regions in Table 1).

Within the disease interval no genes previously associated with EDS were found. Sixteen positional and functional candidate genes, including TGFBR3, RSPO3, ZNF596, SRP14, CHST14, DNAJC17, PPP1R14D, SPINT1, VPS18, VPS39, SNAP23, LRRC57, PDIA3, MFAP1, GALK2 and AP4E1 were selected for sequencing using the Sanger method, and this resulted in the identification of a homozygous deletion of a single nucleotide in the CHST14 gene in the two We report homozygous frameshift mutations in CHST14, encoding dermatan-4-sulfotransferase 1 (D4ST-1), in two unrelated inbred families from Turkish and Indian origin respectively, presenting a form of EDS, which, up till now, has been referred to as EDS VIB. The clinical phenotype in these patients is characterized by distinct craniofacial, musculoskeletal, cutaneous and ophthalmological abnormalities (Suppl. Table S2). Our patients closely resemble the Pakistani siblings reported by Steinmann and co-workers [START_REF] Steinmann | Ehlers-Danlos syndrome in two siblings with deficient lysyl hydroxylase activity in cultured skin fibroblasts but only mild hydroxylysine deficit in skin[END_REF] with a phenotype of EDS kyphoscoliotic type (EDS VI) but normal LH-1 activity, a condition subsequently referred to as EDS VIB. [START_REF] Steinmann | The Ehlers-Danlos Syndrome[END_REF] Loss-of-function mutations in CHST14 were recently shown to cause a rare autosomal recessive condition called "adducted thumb clubfoot syndrome" (ATCS). [START_REF] Dundar | Loss of dermatan-4-sulfotransferase 1 function results in adducted thumb-clubfoot syndrome[END_REF] The EDS patients reported here show many features of ATCS including the craniofacial abnormalities, congenital contractures of thumbs and fingers, clubfeet, joint hyperlaxity, wasted build, thin skin and easy bruising. However, they differ from ATCS in the severity of the cutaneous manifestations, especially in the presence of atrophic scars, and also in the more severe kyphoscoliosis and ocular involvement (Suppl.Table S2).

With the identification of a homozygous NM_130468.2:c.145delG (NP_569735.1:p.Val49*) mutation in patient 1 and 2 of this study, which is identical to the mutation reported earlier in two ATCS-sibs [START_REF] Dundar | Loss of dermatan-4-sulfotransferase 1 function results in adducted thumb-clubfoot syndrome[END_REF], we provide evidence that ATCS and EDS VIB form a continuum within the same clinical spectrum.

Furthermore our findings show that the new EDS-variant, recently reported by Kosho et al [START_REF] Kosho | A new Ehlers-Danlos syndrome with craniofacial characteristics, multiple congenital contractures, progressive joint and skin laxity, and multisystem fragility-related manifestations[END_REF], and also shown to be associated with loss-of-function mutations in CHST14 [START_REF] Miyake | Loss-of-function mutations of CHST14 in a new type of Ehlers-Danlos syndrome[END_REF] is situated within the EDS VIB-ATCS spectrum. Deleted: [START_REF] Steinmann | Ehlers-Danlos syndrome in two siblings with deficient lysyl hydroxylase activity in cultured skin fibroblasts but only mild hydroxylysine deficit in skin[END_REF] Deleted: [START_REF] Steinmann | The Ehlers-Danlos Syndrome[END_REF] Deleted: [START_REF] Dundar | Loss of dermatan-4-sulfotransferase 1 function results in adducted thumb-clubfoot syndrome[END_REF] Deleted: Supplementary Hitherto reported CHST14 mutations comprise six missense mutations, one frameshift mutation and one nonsense mutation [START_REF] Dundar | Loss of dermatan-4-sulfotransferase 1 function results in adducted thumb-clubfoot syndrome[END_REF]. We expand the range of mutations in CHST14

with a novel homozygous 20-bp duplication in the Indian patient (patient 3).

The CHST14-related EDS VIB phenotype is distinguished from EDS VIA by the presence of distinct craniofacial abnormalities, contractures of fingers and toes, tapering fingers, severe wrinkling of the palms, and gastro-intestinal and genito-urinary manifestations (Suppl. Table S2). On the other hand, arterial rupture and rupture of the eye globe, sometimes observed in EDS VIA, have not been observed in patients with CHST14 mutations so far. In order to clearly distinguish the CHST14-related phenotype and its associated risks from the kyphoscoliotic type of EDS, we propose to coin the CHST14-related condition "musculo-contractural Ehlers-Danlos syndrome".

Musculo-contractural EDS shows phenotypic resemblance to the spondylocheirodysplastic form of EDS (SCD-EDS) (Suppl. Table S2), caused by mutations in the SLC39A13, encoding a membrane-bound zinc transporter and in which moderately increased urinary LP/HP ratios (intermediate values between EDS VIA & VIB) have been reported [START_REF] Giunta | Spondylocheiro dysplastic form of the Ehlers-Formatted: Line spacing: Double Field Code Changed Formatted: English (U.K.) Formatted: German[END_REF].

CHST14 encodes the enzyme D4ST-1, which is involved in the biosynthesis of the glycosaminoglycan (GAG) dermatan sulfate (DS), where it catalyzes 4-O-sulfation of N-acetylgalactosamine (GalNAc) [START_REF] Penc | Dermatan sulfate released after injury is a potent promoter of fibroblast growth factor-2 function[END_REF]. GAGs, such as DS and chondroitin sulfate (CS) are long unbranched polymers, whose synthesis starts with the formation of a tetrasaccharide linker region that attaches the GAG chains to a serine residue within the conserved attachment sites of core proteins (Suppl. Fig S1). CS/DS chains are composed of alternating glucuronic acid (GlcA)

and GalNAc disaccharide units. [START_REF] Trowbridge | Dermatan sulfate: new functions from an old glycosaminoglycan[END_REF], In DS, GlcA is epimerized to iduronic acid (IdoA) [START_REF] Maccarana | Biosynthesis of dermatan sulfate: chondroitin-glucuronate C5epimerase is identical to SART2[END_REF][START_REF] Pacheco | Two dermatan sulfate epimerases form iduronic acid domains in dermatan sulfate[END_REF]. Subsequent addition of Deleted: [START_REF] Dundar | Loss of dermatan-4-sulfotransferase 1 function results in adducted thumb-clubfoot syndrome[END_REF]Miyake, et al., 2010) Deleted: Supplementary Deleted: EDS VIB Deleted: Supplementary Deleted: [START_REF] Giunta | Spondylocheiro dysplastic form of the Ehlers-Formatted: Line spacing: Double Field Code Changed Formatted: English (U.K.) Formatted: German[END_REF] Deleted: [START_REF] Penc | Dermatan sulfate released after injury is a potent promoter of fibroblast growth factor-2 function[END_REF] Deleted: ementary Deleted: [START_REF] Maccarana | Biosynthesis of dermatan sulfate: chondroitin-glucuronate C5epimerase is identical to SART2[END_REF][START_REF] Pacheco | Two dermatan sulfate epimerases form iduronic acid domains in dermatan sulfate[END_REF]) [START_REF] Evers | Molecular cloning and characterization of a dermatan-specific N-acetylgalactosamine 4-O-sulfotransferase[END_REF][START_REF] Hiraoka | Molecular cloning and expression of two distinct human N-acetylgalactosamine 4-O-sulfotransferases that transfer sulfate to GalNAc beta 1-->4GlcNAc beta 1-->R in both N-and O-glycans[END_REF][START_REF] Mikami | Specificities of three distinct human chondroitin/dermatan N-acetylgalactosamine 4-O-sulfotransferases demonstrated using partially desulfated dermatan sulfate as an acceptor: implication of differential roles in dermatan sulfate biosynthesis[END_REF] The epimerisation and sulfation reactions are not random, but reflect a tightly controlled system which produces structural variability into the CS/DS chain, thereby determining functional interactions with potential protein partners. [START_REF] Trowbridge | Dermatan sulfate: new functions from an old glycosaminoglycan[END_REF]. Both Dundar et al and Miyake et al showed that the CHST14 mutations result in loss of D4ST-1 activity. Although patients' fibroblasts are able to epimerize GlcA-GalNAc to IdoA-GalNAc, they cannot add sulfate to the C-4 hydroxyl group of IdoA-GalNAc, thereby allowing back-epimerisation from

F
IdoA to GlcA to form CS. [START_REF] Dundar | Loss of dermatan-4-sulfotransferase 1 function results in adducted thumb-clubfoot syndrome[END_REF][START_REF] Miyake | Loss-of-function mutations of CHST14 in a new type of Ehlers-Danlos syndrome[END_REF] This suggests that D4ST-1 is the key enzyme for 4-O-sulfation of DS in skin, which cannot be compensated for by the chondroitin 4-O-sulfotransferases.

DS is covalently attached via an O-xylose linkage to serine residues of core proteins to form DS proteoglycans (DSPG), such as thrombomodulin, versican, and the small leucine-rich proteoglycans (SLRP) decorin and biglycan. These DSPG have widespread tissue distribution in blood vessel walls, cornea, skin, tendon, cartilage, bone and undifferentiated mesenchymal tissue. [START_REF] Trowbridge | Dermatan sulfate: new functions from an old glycosaminoglycan[END_REF] They are involved in organization of the extracellular matrix, wound repair, anti-coagulant processes, cell adhesion, migration and proliferation.

Miyake et al showed that DS of decorin is completely lost and replaced by CS in the patients' fibroblasts. [START_REF] Miyake | Loss-of-function mutations of CHST14 in a new type of Ehlers-Danlos syndrome[END_REF] Decorin is essential in maintaining skin integrity and collagen structure. The protein core of decorin binds to collagen fibrils, and its single DS chain binds to Tenascin-X, another extracellular matrix (ECM) protein which co-localizes with collagen fibrils. Deleted: [START_REF] Evers | Molecular cloning and characterization of a dermatan-specific N-acetylgalactosamine 4-O-sulfotransferase[END_REF][START_REF] Hiraoka | Molecular cloning and expression of two distinct human N-acetylgalactosamine 4-O-sulfotransferases that transfer sulfate to GalNAc beta 1-->4GlcNAc beta 1-->R in both N-and O-glycans[END_REF][START_REF] Mikami | Specificities of three distinct human chondroitin/dermatan N-acetylgalactosamine 4-O-sulfotransferases demonstrated using partially desulfated dermatan sulfate as an acceptor: implication of differential roles in dermatan sulfate biosynthesis[END_REF] Deleted: [START_REF] Dundar | Loss of dermatan-4-sulfotransferase 1 function results in adducted thumb-clubfoot syndrome[END_REF]Miyake, et al., 2010) Deleted: (Miyake, et al., 2010)

F o r P e e r R e v i e w 15
Patients deficient in tenascin-X and mice deficient in decorin both have increased skin fragility and altered skin collagen fibril architecture [START_REF] Danielson | Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility[END_REF][START_REF] Schalkwijk | A recessive form of the Ehlers-Danlos syndrome caused by tenascin-X deficiency[END_REF].

Miyake et al argue that loss of the decorin hybrid CS/DS chain and replacement by CS decreases the flexibility of the GAG chain. This hybrid CS/DS regulates the space between collagen fibrils, and its replacement by CS results in disturbed collagen bundle organisation, as observed on ultrastructural examination of the skin from D4ST-1 deficient patients [START_REF] Miyake | Loss-of-function mutations of CHST14 in a new type of Ehlers-Danlos syndrome[END_REF] and this study).

DSPG bind to a diverse range of extracellular matrix proteins either through their core protein or through their GAG chains. Besides structural molecules such as collagens and tenascin-X, these include growth factors, cytokines, chemokines and protease inhibitors, including proteins involved in the coagulation cascade [START_REF] Trowbridge | Dermatan sulfate: new functions from an old glycosaminoglycan[END_REF]. The broad array of physiological events in which DS and DSPG play a role, and the essential role of D4ST-1 in the biosynthesis of these DS, indicate that altered interaction of these proteoglycans, and disturbed ECM homeostasis underly the pathogenesis of both musculo-contractural EDS and ATCS, and explains the observed multisystemic clinical manifestations which result from disturbances in diverse physiological processes, including development, extracellular matrix maintenance and wound repair. J-L: Thin and hyperextensible skin (J) and cigarette paperscars in patient 2 (K) and 3 (L). Synthesis of CS and DS starts with the formation of a tetrasaccharide linker region that attaches the GAG chains to a serine residue within the conserved attachment sites of core proteins. The activity of Beta4 GalNac Transferase I, that transfers the first GalNAc residue onto the tetrasaccharide linker, starts a growing CS/DS chain, composed of alternating GlcA and GalNAc disaccharide units. [START_REF] Trowbridge | Dermatan sulfate: new functions from an old glycosaminoglycan[END_REF]. In DS, GlcA is epimerised to IdoA, followed by sulfate addition to C-4 hydroxylgroup of the adjacent GalNAc residue by D4ST-1, thereby generating DS from CS. Loss of D4ST-1 activity will result in loss of this 4-O-sulfation lock, and will allow back epimerisation of IdoA to GlcA, replacing DS by CS.

  illustrated in Fig 1.

Family

  J&K) and marked generalized hirsutism. She had tapering fingers, clinodactyly, small joint hypermobility, wrinkled palms (Fig 1E) and dislocation of the radio-ulnar joints (Fig 1I). Her feet were small and broad with short toes, and markedly soft and puffy subcutaneous tissues (Fig 1G). Follow-up examination at age 21 yrs (Fig 1B) revealed moderate thoracolumbar scoliosis with high Deleted: ' on X-rays, absence of breast development and a mild hearing impairment. Echocardiography at the age of 14 yrs was normal with an aortic root of 18 mm. Family 2 (patient 3) Patient 3 is a 12-year old girl and first child of a consanguineous couple of Indian origin. She was born at term after an uneventful pregnancy and delivery with a birth weight of 2800 g (-1.88 SD). Birth length and OFC were not recorded. She presented clubfeet at birth, which were corrected at the age of 18 months. Her fingers and toes were tapering with flexion contractures of all fingers (Fig 1F) and toes (Fig 1H) and flexion-adduction contractures of the thumbs (Fig 1F). She had prominent wrinkles on the palms, but absence of deep palmar creases. At the age of 5 months she developed a kyphoscoliosis which worsened progressively during childhood. Gross motor development was delayed, she had a slender build with low muscle mass and a history of dislocation of the left elbow and tendon injury of the right knee following a fall. Her skin was soft and hyperextensible, especially over elbows and neck. Wound healing was delayed with large gaping wounds and severe atrophic cigarette paperscars (Fig 1L), which needed multiple surgical suturing over knees. She presented easy bruisability and chronic ulcers. Her facial appearance was characterized by blue sclerae, microcornea, prominent bulging eyes, myopia and low-set ears (Fig 1C). At the age of 8 yrs she had a subacute duodenal obstruction due to malrotation, which was treated surgically.

  in the interstitial area, which in addition contained large stellate globules of hyaluronic acid (not shown) . The elastic fibers presented normal ultrastructural features in papillary and reticular dermis (not shown). Fibroblasts exhibited an elongated and/or dilated endoplasmic reticulum (ER) (Fig 2).

FormattedFormatted

  family 1 (NM_130468.2:c.145delG, NP_569735.1:p.Val49*) (Fig 3A). Subsequent sequencing analysis of CHST14 identified a homozygous 20-bp duplication in patient 3 (NM_130468.2:c.981_1000dup, NP_569735.1:p.Glu334Glyfs*107 ) (Fig 3B). The mutations were found in homozygous state only in the affected patients, and were present in the heterozygous state in their parents. The unaffected sister from family 1 has two wild-type alleles. The mutations were not detected in 109 control individuals (218 alleles). Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence (GenBank reference sequence NM_130468.3), according to the journal guidelines. The initiation codon is codon 1.

  C-4 hydroxyl group of GalNAC prevents further epimerization of the adjacent GlcA or IdoA.(Malmstrom, 1984) GalNAc can be sulfated in the 4-O-position by the three major sulfotransferases C4ST-1 and -2 (chondroitin-4-sulfotransferase 1 & 2) and D4ST-1, which have different substrate specificities.

  Overstreet MA, Willing MC, Marini JC, Cabral WA, Pals G, Bristow J, Atsawasuwan P, Yamauchi M, Yeowell HN. 2004. Heterogeneous basis of the type VIB form of Ehlers-Danlos syndrome (EDS VIB) that is unrelated to decreased collagen lysyl hydroxylation. Am J Med Genet A 131(2):155-62. Yeowell H, Steinmann B. 2000. Ehlers-Danlos syndrome, kyphoscoliotic form. Seatlle: University of Washington, Seatlle. Yeowell HN, Walker LC. 2000. Mutations in the lysyl hydroxylase 1 gene that result in enzyme deficiency and the clinical phenotype of Ehlers-Danlos syndrome type VI. Mol Genet Metab 71(1-2):212-24.

Figure 1 :

 1 Figure 1: Clinical characteristics.

Figure 2 :

 2 Figure 2: electron microscopic findings in skin of patient 2. Fibroblasts show dilatation of endoplasmic reticulum. .

Figure 3 :

 3 Figure 3: CHST14 mutations in the patients. A: Pedigree of family 1. Sequence analysis reveals a homozygous G deletion at cDNA position 145 of the CHST14 gene in both affected siblings. The deleted nucleotides are boxed in the WT sequence (upper panel), while the arrow indicates the position of the deleted nucleotide in the mutant sequence (lower panel). B: Pedigree of family 2. Sequence analysis reveals a homozygous 20-bp duplication at cDNA position c.981-1000 of the CHST14 gene in the affected patient. The duplicated nucleotides are boxed in upper and lower panel.

Formatted

  : Clinical pictures of patient 2 at age 14 yrs (A) and age 21 yrs (B), showing craniofacial dysmorphism with flattened profile, malar hypoplasia, bushy eyebrows and synophrys, downslanting palpebral fissures, blue sclerae, microcornea, long philtrum with thin upperlip, and protruding jaw with pointed chin. Clinical picture of patient 3 at age 12 yrs (C). D-I: Hands and feet with characteristic features. Patient 1 at age 22 yrs (D), patient 2 at age 14 (I) and age 21 yrs (E, G) and patient 3 at age 12 yrs (F, H): tapering fingers with flexion contractures and excessive wrinkling of the palms (D-F), foot deformities with short broad feet and toes in patient 2 (G) and long tapering toes with contractures, pseudotumor and cigarette paper scarring in patient 3 (H); dislocation of radio-ulnar joint in patient 2 (I). J-L: Thin and hyperextensible skin (J) and cigarette paperscars in patient 2 (K) and 3 (L). 32x36mm (600 x 600 DPI) the patients. A: Pedigree of family 1. Sequence analysis reveals a homozygous G deletion at cDNA position 145 of the CHST14 gene in both affected siblings. The deleted nucleotides are boxed in the WT sequence (upper panel), while the arrow indicates the position of the deleted nucleotide in the mutant sequence (lower panel). B: Pedigree of family 2. Sequence analysis reveals a homozygous 20-bp duplication at cDNA position c.981-1000 of the CHST14 gene in the affected patient. The duplicated nucleotides are boxed in upper and lower panel.

Table Legend Table 1 :

 Legend1 Candidate regions for family 1 following homozygosity mapping.Suppl. FigureS1: Biosynthesis of Chondroitin and Dermatan Sulfate.

			Human Mutation		Page 26 of 34
	Chr	SNP delineation	#Mb	# genes	# microRNAs
	1 6 8 15 Total	rs284180-rs1325433 rs2875742-rs4371861 rs10488368-rs1822243 rs2412456-rs7176579 F o r P e e r R e v i e w 0.65 10 0 2.31 16 1 3.01 18 1 11.17 173 3 17.14 Mb 217 5
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	Page 33 of 34				Human Mutation				Page
			muscles				Diassasis recti		broadening
			diastasis recti				(1/7)		Thenar
							Contractures		hypotrophy
							elbows/knees
							(1/7)
	OTHER Hearing impairment Intestinal Genito-urinary	+ Abdominal cramping nephrolithiasis Nephrolithias + Abdominal cramping is-renal ptosis with ureteral obstruction F o r P e e r R e v i e w --+ (4/6) + malrotation with duodenal obstruction -Constipation (5/6) Diverticula perforation (1/6) Absence of gastrocolic omentum, spontaneous volvulus of small intestine (1/7) -Nephrolithiasis and hydronephrosis retention (1/7) (1/2) Nephrolithiasis (1/6) Urinary Hydronephrosis (2/7) Nephrolithiasis Coarctatio aortae (1/7) † (5/11)	---	---
						Cryptorchidism	Horseshoekidney
						(1/2M)	(1/7)
							Crytorchidism
							(4/4 M)
	Other	-	Rupture	-	† (1/2)	Diastasis recti	Cleft lip/ palate	-	Platyspondyly
			abdominal			(1/6)	(2/7)		Metaphyseal
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Acknowledgments:

We wish to thank the patients and their parents for participating in this work. FM, DS and PV are fellows of the Fund for Scientific Research (FWO), Flanders, Belgium. This work was supported by a Methusalem Grant 08/01M01108 from the Ghent University to ADP and Grant G.0171.05 from the Fund for Scientific Research (FWO), Flanders, Belgium to ADP.

Field Code Changed

Field Code Changed Field Code Changed Deleted: (Danielson, et al., 1997;Schalkwijk, et al., 2001) Deleted: (Miyake, et al., 2010 Deleted: EDS VIB

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Human Mutation

F o r P e e r R e v i e w

Suppl. Table S1: CHST14 sequencing oligonucleotide primer pairs. Fragments 1 to 4 amplify the coding region whereas fragments 5 and 6 amplify the 3'UTR of the gene. For mix A, PCR amplification was performed with 50ng genomic DNA, 0.6 µM of each primer (oligonucleotide primer pairs (Invitrogen, Carlsbad, CA, USA) are listed in Supplementary Table 1), 0.12 mM dNTP mix (Invitrogen), and 1 unit of KAPA2G Robust HotStart DNA polymerase in 1 x KAPA2G Robust HotStart GC Buffer (Kapa Biosystems, Woburn, MA, USA).

For mix B, PCR amplification was performed with 50ng genomic DNA, 0.6 µM of each primer (oligonucleotide primer pairs (Invitrogen) are listed in Supplementary Table 1), 1 mM MgCl 2 , 0.12 mM dNTP mix (Invitrogen), and 0.5 units of KAPATaq HotStart DNA polymerase in 1 x KAPATaq HotStart Buffer (Kapa Biosystems, Woburn, MA, USA).

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F o r P e e r R e v i e w

For TD62-50 thermal cycling conditions consisted of an initial denaturation step of 94°C for 4 minutes, 12 cycles of 94°C for 20s, 62°C for 15s, and 72°C for 60s with a decrease in annealing temperature of 1°C each cycle, followed by 24 cycles of 94°C for 40s, 50°C for 40s, and 72°C for 30s, and a final extension at 72°C for 10 minutes.

For AT60 thermal cycling conditions consisted of an initial denaturation step of 94°C for 4 minutes, 32 cycles of 94°C for 20s, 60°C for 20s, and 72°C for 40s followed by a final extension at 72°C for 10 minutes.

Suppl. Table 2:

Summary of the clinical findings in the currently reported patients, and comparison with patients reported by Steinmann et al [START_REF] Steinmann | Ehlers-Danlos syndrome in two siblings with deficient lysyl hydroxylase activity in cultured skin fibroblasts but only mild hydroxylysine deficit in skin[END_REF][START_REF] Steinmann | The Ehlers-Danlos Syndrome[END_REF], Kosho et al (Kosho, et al., 2010), patients with typical EDS VIA, patients with ATCS [START_REF] Dundar | Loss of dermatan-4-sulfotransferase 1 function results in adducted thumb-clubfoot syndrome[END_REF] and patients with spondylocheirodysplastic (SCD) EDS [START_REF] Giunta | Spondylocheiro dysplastic form of the Ehlers-Formatted: Line spacing: Double Field Code Changed Formatted: English (U.K.) Formatted: German[END_REF].

Abbreviations