The epitheliogenesis imperfecta locus maps to equine chromosome 8 in American Saddlebred horses

Epitheliogenesis imperfecta (EI) is a hereditary junctional mechanobullous disease that occurs in newborn American Saddlebred foals. The pathological signs of epitheliogenesis imperfecta closely match a similar disease in humans known as Herlitz junctional epidermolysis bullosa, which is caused by a mutation in one of the genes (LAMA3, LAMB3 and LAMC2) coding for the subunits of the laminin 5 protein (laminin alpha3, laminin beta3 and laminin gamma2). The LAMA3 gene has been assigned to equine chromosome 8 and LAMB3 and LAMC2 have been mapped to equine chromosome 5. Linkage disequilibrium between microsatellite markers that mapped to equine chromosome 5 and equine chromosome 8 and the EI disease locus was tested in American Saddlebred horses. The allele frequencies of microsatellite alleles at 11 loci were determined for both epitheliogenesis imperfecta affected and unaffected populations of American Saddlebred horses by genotyping and direct counting of alleles. These were used to determine fit to Hardy-Weinberg equilibrium for control and EI populations using Chi square analysis. Two microsatellite loci located on equine chromosome 8q, ASB14 and AHT3, were not in Hardy-Weinberg equilibrium in affected American Saddlebred horses. In comparison, all of the microsatellite markers located on equine chromosome 5 were in Hardy-Weinberg equilibrium in affected American Saddlebred horses. This suggested that the EI disease locus was located on equine chromosome 8q, where LAMA3 is also located.     

A frameshift mutation within LAMC2 is responsible for Herlitz type junctional epidermolysis bullosa (HJEB) in black headed mutton sheep

Junctional epidermolysis bullosa (JEB) is a hereditary mechanobullous skin disease in humans and animals. A Herlitz type JEB was identified in German Black Headed Mutton (BHM) sheep and affected lambs were reproduced in a breeding trial. Affected lambs showed skin and mucous membranes blistering and all affected lambs died within the first weeks of life. The pedigree data were consistent with a monogenic autosomal recessive inheritance. Immunofluorescence showed a reduced expression of laminin 5 protein which consists of 3 subunits encoded by the genes LAMA3, LAMB3 and LAMC2. We screened these genes for polymorphisms. Linkage and genome-wide association analyses identified LAMC2 as the most likely candidate for HJEB. A two base pair deletion within exon 18 of the LAMC2 gene (FM872310:c.2746delCA) causes a frameshift mutation resulting in a premature stop codon (p.A928*) 13 triplets downstream of this mutation and in addition, introduces an alternative splicing of exon 18 LAMC2. This deletion showed a perfect co-segregation with HJEB in all 740 analysed BHM sheep. Identification of the LAMC2 deletion means an animal model for HJEB is now available to develop therapeutic approaches of relevance to the human form of this disease.     

Laminin 5 mutations in junctional epidermolysis bullosa: molecular basis of Herlitz vs non-Herlitz phenotypes

Junctional epidermolysis bullosa (JEB) is a group of heritable blistering diseases in which tissue separation occurs within the lamina lucida of the cutaneous basement membrane zone. Clinically, two broad subcategories have been recognized: The Herlitz variant (H-JEB; OMIM 226700) is characterized by early demise of the affected individuals, usually within the first year of life, while non-Herlitz (nH-JEB; OMIM 226650) patients show a milder phenotype with life-long blistering, yet with normal lifespan. In this study, we have examined a cohort of 27 families, 15 with Herlitz and 12 with non-Herlitz JEB, for mutations in the candidate genes, LAMA3, LAMB3, and LAMC2, encoding the subunit polypeptides of laminin 5. The mutation detection strategy consisted of PCR amplification of all exons in these genes, followed by heteroduplex scanning and nucleotide sequencing. We were able to identify pathogenic mutations in both alleles of each proband, the majority of the mutations being in the LAMB3 gene. Examination of the mutation database revealed that most cases with Herlitz JEB harbored premature termination codon (PTC) mutations in both alleles. In non-Herlitz cases, the PTC mutation was frequently associated with a missense mutation or a putative splicing mutation in trans. In three cases with putative splicing mutations, RT-PCR analysis revealed a repertoire of splice variants in-frame, predicting the synthesis of either shortened or lengthened, yet partly functional, polypeptides. These observations would explain the relatively mild phenotype in cases with splicing mutations. Collectively, these findings, together with the global laminin 5 mutation database, contribute to our understanding of the genotype/phenotype correlations explaining the Herlitz vs non-Herlitz phenotypes.     

Laminin 332 in junctional epidermolysis bullosa

Laminin 332 is an essential component of the dermal-epidermal junction, a highly specialized basement membrane zone that attaches the epidermis to the dermis and thereby provides skin integrity and resistance to external mechanical forces. Mutations in the LAMA3, LAMB3 and LAMC2 genes that encode the three constituent polypeptide chains, α3, β3 and γ2, abrogate or perturb the functions of laminin 332. The phenotypic consequences are diminished dermal-epidermal adhesion and, as clinical symptoms, skin fragility and mechanically induced blistering. The disorder is designated as junctional epidermolysis bullosa (JEB). This article delineates the signs and symptoms of the different forms of JEB, the mutational spectrum, genotype-phenotype correlations as well as perspectives for future molecular therapies.     

A mutation in the LAMC2 gene causes the Herlitz junctional epidermolysis bullosa (H-JEB) in two French draft horse breeds

Epidermolysis bullosa (EB) is a heterogeneous group of inherited diseases characterised by skin blistering and fragility. In humans, one of the most severe forms of EB known as Herlitz-junctional EB (H-JEB), is caused by mutations in the laminin 5 genes. EB has been described in several species, like cattle, sheep, dogs, cats and horses where the mutation, a cytosine insertion in exon 10 of the LAMC2 gene, was very recently identified in Belgian horses as the mutation responsible for JEB. In this study, the same mutation was found to be totally associated with the JEB phenotype in two French draft horse breeds, Trait Breton and Trait Comtois. This result provides breeders a molecular test to better manage their breeding strategies by genetic counselling.     

cDNA sequence of the horse (Equus caballus) LAMA3 gene and characterization of two intronic SNP markers.

Laminins are large heterotrimeric basement membrane glycoproteins composed of alpha, beta and gamma chains. The Laminin 5 isoform has an alpha3beta3gamma2 composition and is essential for the adhesion of basal keratinocytes to the underlying epithelial basement membrane where it is mainly located. Mutations in the genes coding for the 3 chains have been associated with a severe skin blistering disease, Herlitz's junctional epidermolysis bullosa (JEB), observed in different species as man, dog, cat and horse. In this study, we report the sequence of the 5.2 kb horse laminin alpha 3 cDNA (LAMA3) as well as the detection of two intronic SNPs. These data will be useful to further identify causal mutations for the disease in this gene.     

Two Novel Mutations in LAMC2 Gene in Iranian Families Affected by Junctional Epidermolysis Bullosa

Background:Junctional epidermolysis bullosa (JEB) is an autosomal recessive skin disorder with defective adhesion of dermal- epidermal within the lamina lucida region of the basement membrane zone. The main characterization of JEB is blistering and fragile skin and mucous membrane. Laminins are noncollagenous part of basement membrane and classified as a family of extracellular matrix glycoprotein. Laminins contain three chains: Laminin α, Laminin β and Laminin γ. LAMC2 (laminin subunit gamma 2) gene encodes γ subunit of laminin and its mutation contributes to JEB. Here, we report a disease-causing nonsense mutation and a large deletion mutation in LAMC2 gene in two families affected by JEB.Methods:Whole exome sequencing (WES) was carried out on the mother of patient in family I and the patient himself in family II to detect the underlying mutations. Then, sanger sequencing was performed to confirm the identified mutations.Results:Next generation sequencing (NGS) data analysis of the first family showed a novel, nonsense mutation in LAMC2 gene (LAMC2: {"type":"entrez-nucleotide","attrs":{"text":"NM_005562","term_id":"1653960840"}}NM_005562: exon14:c.C2143T: p.R715X). The heterozygous state of the mutation was confirmed by sanger sequencing in the parents and unaffected brother. In Family II, NGS data had no coverage in the large area of LAMC2 gene. Thus, to confirm the possible deletion sanger sequencing was done and blasting of sequence showed the deleted region of 9.4 kb (exon10-17) in LAMC2 gene.Conclusion:In summary, current study reported a novel disease-causing premature termination codon (PTC) mutation in LAMC2 gene and a large deletion mutation in patients affected by JEB.Key Words: Junctional Epidermolysis Bullosa, LAMC2 gene, Novel mutation, Skin disorder     

Novel sequence variations in LAMA2 andSGCG genes modulating cis-acting regulatory elements and RNA secondary structure

In this study, we detected new sequence variations in LAMA2 and SGCG genes in 5 ethnic populations, and analysed their effect on enhancer composition and mRNA structure. PCR amplification and DNA sequencing were performed and followed by bioinformatics analyses using ESEfinder as well as MFOLD software. We found 3 novel sequence variations in the LAMA2 (c.3174+22_23insAT and c.6085 +12delA) and SGCG (c. (*) 102A/C) genes. These variations were present in 210 tested healthy controls from Tunisian, Moroccan, Algerian, Lebanese and French populations suggesting that they represent novel polymorphisms within LAMA2 and SGCG genes sequences. ESEfinder showed that the c. (*) 102A/C substitution created a new exon splicing enhancer in the 3'UTR of SGCG genes, whereas the c.6085 +12delA deletion was situated in the base pairing region between LAMA2 mRNA and the U1snRNA spliceosomal components. The RNA structure analyses showed that both variations modulated RNA secondary structure. Our results are suggestive of correlations between mRNA folding and the recruitment of spliceosomal components mediating splicing, including SR proteins. The contribution of common sequence variations to mRNA structural and functional diversity will contribute to a better study of gene expression.     

Combining GWAS and RNA-Seq Approaches for Detection of the Causal Mutation for Hereditary Junctional Epidermolysis Bullosa in Sheep

In this study, we demonstrate the use of a genome-wide association mapping together with RNA-seq in a reduced number of samples, as an efficient approach to detect the causal mutation for a Mendelian disease. Junctional epidermolysis bullosa is a recessive genodermatosis that manifests with neonatal mechanical fragility of the skin, blistering confined to the lamina lucida of the basement membrane and severe alteration of the hemidesmosomal junctions. In Spanish Churra sheep, junctional epidermolysis bullosa (JEB) has been detected in two commercial flocks. The JEB locus was mapped to Ovis aries chromosome 11 by GWAS and subsequently fine-mapped to an 868-kb homozygous segment using the identical-by-descent method. The ITGB4, which is located within this region, was identified as the best positional and functional candidate gene. The RNA-seq variant analysis enabled us to discover a 4-bp deletion within exon 33 of the ITGB4 gene (c.4412_4415del). The c.4412_4415del mutation causes a frameshift resulting in a premature stop codon at position 1472 of the integrin β4 protein. A functional analysis of this deletion revealed decreased levels of mRNA in JEB skin samples and the absence of integrin β4 labeling in immunohistochemical assays. Genotyping of c.4412_4415del showed perfect concordance with the recessive mode of the disease phenotype. Selection against this causal mutation will now be used to solve the problem of JEB in flocks of Churra sheep. Furthermore, the identification of the ITGB4 mutation means that affected sheep can be used as a large mammal animal model for the human form of epidermolysis bullosa with aplasia cutis. Our approach evidences that RNA-seq offers cost-effective alternative to identify variants in the species in which high resolution exome-sequencing is not straightforward.     

Digenic junctional epidermolysis bullosa: mutations in COL17A1 and LAMB3 genes

Junctional epidermolysis bullosa (JEB), a genetically heterogeneous group of blistering skin diseases, can be caused by mutations in the genes encoding laminin 5 or collagen XVII, which are components of the hemidesmosome-anchoring filament complex in the skin. Here, a family with severe nonlethal JEB and with mutations in genes for both proteins was identified. The index patient was compound heterozygous for the COL17A1 mutations L855X and R1226X and was heterozygous for the LAMB3 mutation R635X. As a consequence, two functionally related proteins were affected. Absence of collagen XVII and attenuated laminin 5 expression resulted in rudimentary hemidesmosome structure and separation of the epidermis from the basement membrane, with severe skin blistering as the clinical manifestation. In contrast, single heterozygotes carrying either (1) one or the other of the COL17A1 null alleles or (2) a double heterozygote for a COL17A1 and a LAMB3 null allele did not have a pathological skin phenotype. These observations indicate that the known allelic heterogeneity in JEB is further complicated by interactions between unlinked mutations. They also demonstrate that identification of one mutation in one gene is not sufficient for determination of the genetic basis of JEB in a given family.     

Detection of novel LAMC2 mutations in Herlitz junctional epidermolysis Bullosa.

BACKGROUND: Laminin 5, an anchoring filament attachment protein within the lamina lucida of the basement membrane zone involved in the pathogenesis of junctional epidermolysis bullosa (JEB), consists of three polypeptide subunits, the alpha 3, beta 3, and gamma 2 chains which are encoded by the LAMA3, LAMB3, and LAMC2 genes, respectively. To facilitate identification of pathogenetic mutations in LAMC2, a strategy based on direct amplification of genomic DNA by PCR or mRNA by RT-PCR, followed by heteroduplex analysis of the PCR products, was developed. MATERIALS AND METHODS: Primer pairs for amplification of the complete cDNA as well as the 23 individual exons in the genomic DNA, which encode the entire gamma 2 chain of laminin 5, were established. The primers for amplification of exons from genomic DNA were positioned at least 24 bp away from the intron-exon borders in the flanking intronic sequences. For amplification of cDNA generated by RT-PCR, eight primer pairs covering overlapping segments of the entire coding sequence of LAMC2 mRNA were used. The amplified sequences were scanned for pathogenetic mutations and sequence variations in JEB patients and unrelated control individuals by heteroduplex analysis by means of conformation sensitive gel electrophoresis (CSGE). RESULTS: Utilizing the strategy developed in this study, we identified pathogenetic mutations in three patients with the Herlitz (lethal) variant of JEB, and eight intragenic normal polymorphisms, which are useful for linkage analysis, in the LAMC2 gene. CONCLUSIONS: The methodology described in this study is capable of detecting single-base substitutions or small insertions and deletions in the LAMC2 gene. Demonstration of mutations in this gene in JEB patients further emphasizes the role of laminin 5 in providing integrity to the cutaneous basement membrane zone.     

Genetics of swayback in American Saddlebred horses

Extreme lordosis, also called swayback, lowback or softback, can occur as a congenital trait or as a degenerative trait associated with ageing. In this study, the hereditary aspect of congenital swayback was investigated using whole genome association studies of 20 affected and 20 unaffected American Saddlebred (ASB) Horses for 48,165 single-nucleotide polymorphisms (SNPs). A statistically significant association was identified on ECA20 (corrected P=0.017) for SNP BIEC2-532523. Of the 20 affected horses, 17 were homozygous for this SNP when compared to seven homozygotes among the unaffected horses, suggesting a major gene with a recessive mode of inheritance. The result was confirmed by testing an additional 13 affected horses and 166 unaffected horses using 35 SNPs in this region of ECA20 (corrected P=0.036). Combined results for 33 affected horses and 287 non-affected horses allowed identification of a region of homozygosity defined by four SNPs in the region. Based on the haplotype defined by these SNPs, 80% of the 33 affected horses were homozygous, 21% heterozygous and 9% did not possess the haplotype. Among the non-affected horses, 15% were homozygous, 47% heterozygous and 38% did not possess the haplotype. The differences between the two groups were highly significant (P<0.00001). The region defined by this haplotype includes 53 known and predicted genes. Exons from three candidate genes, TRERF1, RUNX2 and CNPY3 were sequenced without finding distinguishing SNPs. The mutation responsible for swayback may lie in other genes or in regulatory regions outside exons. This information can be used by breeders to reduce the occurrence of swayback among their livestock. This condition may serve as a model for investigation of congenital skeletal deformities in other species.     

Three Novel Homozygous Point Mutations and a New Polymorphism in the COL17A1 Gene: Relation to Biological and Clinical Phenotypes of Junctional Epidermolysis Bullosa

Junctional epidermolysis bullosa (JEB) is a clinically and biologically heterogeneous genodermatosis, characterized by trauma-induced blistering and healing without scarring but sometimes with skin atrophy. We investigated three unrelated patients with different JEB phenotypes. Patients 1 and 2 had generalized atrophic benign epidermolysis bullosa (GABEB), with features including skin atrophy and alopecia. Patient 3 had the localisata variant of JEB, with predominantly acral blistering and normal hair. All patients carried novel homozygous point mutations (Q1016X, R1226X, and R1303Q) in the COL17A1 gene encoding collagen XVII, a hemidesmosomal transmembrane component; and, therefore, not only GABEB but also the localisata JEB can be a collagen XVII disorder. The nonsense mutations led to drastically reduced collagen XVII mRNA and protein levels. In contrast, the missense mutation allowed expression of abnormal collagen XVII, and epidermal extracts from that patient contained polypeptides of normal size, as well as larger aggregates. The homozygous nonsense mutations in the COL17A1 gene were consistent with the absence of the collagen from the skin and with the GABEB phenotype, whereas homozygosity for the missense mutation resulted in expression of aberrant collagen XVII and, clinically, in localisata JEB.     

Molecular analysis of hereditary nonpolyposis colorectal cancer in the United States: high mutation detection rate among clinically selected families and characterization of an American founder genomic deletion of the MSH2 gene

The identification of germline mutations in families with HNPCC is hampered by genetic heterogeneity and clinical variability. In previous studies, MSH2 and MLH1 mutations were found in approximately two-thirds of the Amsterdam-criteria-positive families and in much lower percentages of the Amsterdam-criteria-negative families. Therefore, a considerable proportion of HNPCC seems not to be accounted for by the major mismatch repair (MMR) genes. Does the latter result from a lack of sensitivity of mutation detection techniques, or do additional genes underlie the remaining cases? In this study we address these questions by thoroughly investigating a cohort of clinically selected North American families with HNPCC. We analyzed 59 clinically well-defined U.S. families with HNPCC for MSH2, MLH1, and MSH6 mutations. To maximize mutation detection, different techniques were employed, including denaturing gradient gel electrophoresis, Southern analysis, microsatellite instability, immunohistochemistry, and monoallelic expression analysis. In 45 (92%) of the 49 Amsterdam-criteria-positive families and in 7 (70%) of the 10 Amsterdam-criteria-negative families, a mutation was detected in one of the three analyzed MMR genes. Forty-nine mutations were in MSH2 or MLH1, and only three were in MSH6. A considerable proportion (27%) of the mutations were genomic rearrangements (12 in MSH2 and 2 in MLH1). Notably, a deletion encompassing exons 1-6 of MSH2 was detected in seven apparently unrelated families (12% of the total cohort) and was subsequently proven to be a founder. Screening of a second U.S. cohort with HNPCC from Ohio allowed the identification of two additional kindreds with the identical founder deletion. In the present study, we show that optimal mutation detection in HNPCC is achieved by combining accurate and expert clinical selection with an extensive mutation detection strategy. Notably, we identified a common North American deletion in MSH2, accounting for approximately 10% of our cohort. Genealogical, molecular, and haplotype studies showed that this deletion represents a North American founder mutation that could be traced back to the 19th century.     

DNA-PKcs mutations in dogs and horses: allele frequency and association with neoplasia

Previously, spontaneous genetic immunodeficiencies in mice, Arabian foals, and recently in Jack Russell terriers have been ascribed to defects in DNA-PKcs (catalytic subunit of the DNA dependent protein kinase) expression. In severe combined immunodeficiency (SCID) foals, a 5 bp deletion at codon 9480 results in a frameshift and a 967 amino acid deletion from the C terminus (including the entire PI3 kinase domain) and an unstable mutant protein. In SCID mice, a single base pair mutation results in a premature stop codon and deletion of 83 amino acids; as in SCID foals, the mutant protein is unstable. Here, we define the mutation within the canine DNA-PKcs gene that results in SCID. In this case, a point mutation results in a stop codon at nucleotide 10,828 and premature termination at a position 517 amino acids before the normal C terminus resulting in a functionally null allele. Thus, this is the third documentation of a spontaneous germline mutation in the C terminus of DNA-PKcs. Emerging data implicate DNA repair factors as potential tumor suppressors. Here, we have ascertained the carrier frequency of the defective DNA-PKcs genes in Arabian horses and in Jack Russell terriers. Our data indicate (in good agreement with a previous report) that the carrier frequency of the equine SCID allele is approximately 8%; in contrast, the carrier frequency of the canine SCID allele is less than 1.1%. We also assessed the frequency of the equine SCID allele in a series of 295 tumors from Arabian horses. We find a statistically significant correlation between the development of a virally induced tumor (sarcoid) and heterozygosity for the equine SCID allele. These data provide further support for an emerging consensus: that DNA-PK may normally act as a tumor suppressor through its caretaker role in maintaining chromosomal stability.     

Novel ENAM and LAMB3 Mutations in Chinese Families with Hypoplastic Amelogenesis Imperfecta

Amelogenesis imperfecta is a group of inherited diseases affecting the quality and quantity of dental enamel. To date, mutations in more than ten genes have been associated with non-syndromic amelogenesis imperfecta (AI). Among these, ENAM and LAMB3 mutations are known to be parts of the etiology of hypoplastic AI in human cases. When both alleles of LAMB3 are defective, it could cause junctional epidermolysis bullosa (JEB), while with only one mutant allele in the C-terminus of LAMB3, it could result in severe hypoplastic AI without skin fragility. We enrolled three Chinese families with hypoplastic autosomal-dominant AI. Despite the diagnosis falling into the same type, the characteristics of their enamel hypoplasia were different. Screening of ENAM and LAMB3 genes was performed by direct sequencing of genomic DNA from blood samples. Disease-causing mutations were identified and perfectly segregated with the enamel defects in three families: a 19-bp insertion mutation in the exon 7 of ENAM (c.406_407insTCAAAAAAGCCGACCACAA, p.K136Ifs*16) in Family 1, a single-base deletion mutation in the exon 5 of ENAM (c. 139delA, p. M47Cfs*11) in Family 2, and a LAMB3 nonsense mutation in the last exon (c.3466C>T, p.Q1156X) in Family 3. Our results suggest that heterozygous mutations in ENAM and LAMB3 genes can cause hypoplastic AI with markedly different phenotypes in Chinese patients. And these findings extend the mutation spectrum of both genes and can be used for mutation screening of AI in the Chinese population.     

Deletion of DNA lying close to the glkA locus induces ectopic sporulation in Streptomyces coelicolor A3(2)

Streptomyces coelicolor A3(2) J1668 sporulated ectopically in the substrate hyphae (the Esp phenotype) with the same time course as sporulation in the aerial hyphae. Examination of related strains implied that the Esp phenotype was caused by the deletion of DNA that lies close to, but is distinct from, the glucose kinase gene ( glkA ). Co-transduction of the Esp phenotype with the deletion present in J1668 confirmed this hypothesis. The size of the deletion was found to be 7.4 kb. Construction of a strain carrying both the J1668 deletion and a whiG mutation demonstrated that the Esp phenotype depends on at least one of the genes required for the differentiation of aerial hyphae into spores.     

Whole-genome sequencing identifies a homozygous deletion encompassing exons 17 to 23 of the integrin beta 4 gene in a Charolais calf with junctional epidermolysis bullosa

Background

Since 2010, four Charolais calves with a congenital mechanobullous skin disorder that were born in the same herd from consanguineous matings were reported to us. Clinical and histopathological examination revealed lesions that are compatible with junctional epidermolysis bullosa (JEB).

Results

Fifty-four extended regions of homozygosity (>1 Mb) were identified after analysing the whole-genome sequencing (WGS) data from the only case available for DNA sampling at the beginning of the study. Filtering of variants located in these regions for (i) homozygous polymorphisms observed in the WGS data from eight healthy Charolais animals and (ii) homozygous or heterozygous polymorphisms found in the genomes of 234 animals from different breeds did not reveal any deleterious candidate SNPs (single nucleotide polymorphisms) or small indels. Subsequent screening for structural variants in candidate genes located in the same regions identified a homozygous deletion that includes exons 17 to 23 of the integrin beta 4 (ITGB4), a gene that was previously associated with the same defect in humans. Genotyping of a second case and of six parents of affected calves (two sires and four dams) revealed a perfect association between this mutation and the assumed genotypes of the individuals. Mining of Illumina BovineSNP50 Beadchip genotyping data from 6870 Charolais cattle detected only 44 heterozygous animals for a 5.6-Mb haplotype around ITGB4 that was shared with the carriers of the mutation. Interestingly, none of the 16 animals genotyped for the deletion carried the mutation, which suggests a rather recent origin for the mutation.

Conclusions

In conclusion, we successfully identified the causative mutation for a very rare autosomal recessive mutation with only one case by exploiting the most recent DNA sequencing technologies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0110-z) contains supplementary material, which is available to authorized users.