Byers Lab: Molecular Genetic Research Interests and Publications

In the last five years, genetic heterogeneity of OI has been established by the identification of mutations in several genes.  Mutations in genes that encode proteins involved in the prolyl-3-hydroxylation pathway of collagen (CRTAP, LEPRE1 and PPIB), as well as genes that encode protein monitors of procollagen modification and processing in the endoplasmic reticulum (PLOD2, FKBP10, SERPINH1) lead to clinical outcomes that mimic the effects of dominant mutations in type I collagen genes.  The shared biochemical and molecular pathways of these new genes are targets for therapeutic intervention and for understanding how abnormal procollagen chains are processed in and outside the cell.  Abstracts of our recent publications follow. 

WNT1 mutations in families affected by moderately severe and progressive recessive osteogenesis imperfecta.  Pyott SM et al.  Am J Hum Genet. 2013 Apr 4;92(4):590-7.

Osteogenesis imperfecta (OI) is a heritable disorder that ranges in severity from death in the perinatal period to an increased lifetime risk of fracture. Mutations in COL1A1 and COL1A2, which encode the chains of type I procollagen, result in dominant forms of OI, and mutations in several other genes result in recessive forms of OI. Here, we describe four recessive-OI-affected families in which we identified causative mutations in wingless-type MMTV integration site family 1 (WNT1). In family 1, we identified a homozygous missense mutation by exome sequencing. In family 2, we identified a homozygous nonsense mutation predicted to produce truncated WNT1. In family 3, we found a nonsense mutation and a single-nucleotide duplication on different alleles, and in family 4, we found a homozygous 14 bp deletion. The mutations in families 3 and 4 are predicted to result in nonsense-mediated mRNA decay and the absence of WNT1. WNT1 is a secreted signaling protein that binds the frizzled receptor (FZD) and the coreceptor low-density lipoprotein-receptor-related protein 5 (LRP5). Biallelic loss-of-function mutations in LRP5 result in recessive osteoporosis-pseudoglioma syndrome with low bone mass, whereas heterozygous gain-of-function mutations result in van Buchem disease with elevated bone density. Biallelic loss-of-function mutations in WNT1 result in a recessive clinical picture that includes bone fragility with a moderately severe and progressive presentation that is not easily distinguished from dominant OI type III.

Allelic background of LEPRE1 mutations that cause recessive forms of osteogenesis imperfecta in different populations.  Pepin MG et al.   Mol Genet Genomic Med 2013 Nov;1(4):194-205.

Biallelic mutations in LEPRE1 result in recessively inherited forms of osteogenesis imperfecta (OI) that are often lethal in the perinatal period. A mutation (c.1080+1G>T, IVS5+1G>T) in African Americans has a carrier frequency of about 1/240. The mutant allele originated in West Africa in tribes of Ghana and Nigeria where the carrier frequencies are 2% and 5%. By examining 200 samples from an African-derived population in Tobago and reviewing hospital neonatal death records, we determined that the carrier frequency of c.1080+1G>T was about one in 200 and did not contribute to the neonatal deaths recorded over a 3-year period of time in Trinidad. In the course of sequence analysis, we found surprisingly high LEPRE1 allelic persity in the Tobago DNA samples in which there were 11 alleles distinguished by a single basepair variant in or near exon 5. All the alleles found in the Tobago population that were within the sequence analysis region were found in the African American population in the Exome Variant Project. This persity appeared to reflect the geographic origin of the original population in Tobago. In 44 inpiduals with biallelic LEPRE1 mutations identified by clinical diagnostic testing, we found the sequence alterations occurred on seven of the 11 variant alleles. All but one of the mutations identified resulted in mRNA or protein instability for the majority of the transcripts from the altered allele. These findings suggest that the milder end of the clinical spectrum could be due to as yet unidentified missense mutations in LEPRE1.

Mutations in PPIB (cyclophilin B) delay type I procollagen chain association and result in perinatal lethal to moderate osteogenesis imperfecta phenotypes.  Pyott SM et al. Human Molec Genet 2011, Apr 15;20(8):1595-609.

Recessive mutations in the cartilage-associated protein (CRTAP), leucine proline-enriched proteoglycan 1 (LEPRE1) and peptidyl prolylcistrans isomerase B (PPIB) genes result in phenotypes that range from lethal in the perinatal period to severe deforming osteogenesis imperfecta (OI). These genes encode CRTAP (encoded by CRTAP), prolyl 3-hydroxylase 1 (P3H1; encoded by LEPRE1) and cyclophilin B (CYPB; encoded by PPIB), which reside in the rough endoplasmic reticulum (RER) and can form a complex involved in prolyl 3-hydroxylation in type I procollagen. CYPB, a prolyl cis–trans isomerase, has been thought to drive the prolyl-containing peptide bonds to the transconfiguration needed for triple helix formation. Here, we describe mutations in PPIB identified in cells from three inpiduals with OI. Cultured dermal fibroblasts from the most severely affected infant make some overmodified type I procollagen molecules. Proα1(I) chains are slow to assemble into trimers, and abnormal procollagen molecules concentrate in the RER, and bind to protein disulfide isomerase (PDI) and prolyl 4-hydroxylase 1 (P4H1). These findings suggest that although CYPB plays a role in helix formation another effect is on folding of the C-terminal propeptide and trimer formation. The extent of procollagen accumulation and PDI/P4H1 binding differs among cells with mutations in PPIBCRTAP and LEPRE1 with the greatest amount in PPIB-deficient cells and the least in LEPRE1-deficient cells. These findings suggest that prolyl cis–trans isomerase may be required to effectively fold the proline-rich regions of the C-terminal propeptide to allow proα chain association and suggest an order of action for CRTAP, P3H1 and CYPB in procollagen biosynthesis and pathogenesis of OI.

Recurrence of perinatal lethal osteogenesis imperfecta in sibships: parsing the risk between parental mosaicism for dominant mutations and autosomal recessive inheritance. Pyott SM, et al. Genet Med 2011 Feb;13(2):125-30.

Recurrence of lethal osteogenesis imperfecta in families results from either dominant (parental mosaicism) or recessive inheritance. The proportion of these two mechanisms is not known, and determination of the contribution of each is important to structure genetic counseling for these families.We measured the recurrence rate of lethal osteogenesis imperfecta after the birth of an affected infant. We determined the rate of parental mosaicism in a subset of families in which we had identified dominant mutations. In 37 families in which two or more affected infants were born, we identified mutations and determined the proportion that resulted from recessive inheritance.The recurrence rate after the first affected pregnancy was 1.3%. The rate of parental mosaicism in families in which a dominant mutation was identified in a first affected child was 16%. In 37 families with two affected infants, 26 had dominant mutations, seven had recessive mutations, and we failed to find mutations in four. The overall recurrence rate for couples after two or more affected infants was 32%; 27% for families with parental mosaicism, 31% for recessive mutations, and 50% for families with no identified mutation.  In most populations, recurrence of lethal osteogenesis imperfecta usually results from parental mosaicism for dominant mutations, but the carrier frequency of recessive forms of osteogenesis imperfecta will alter that proportion. Mutation identification is an important tool to assess risk and facilitate prenatal or preimplantation diagnosis.

Homozygosity for a Missense Mutation in SERPINH1 which Encodes the Collagen Chaperone Protein HSP47,Results in Severe Recessive Osteogenesis Imperfecta .  Christiansen HE et al.  The American Journal of Human Genetics 2010 March 12;86:389-398

Osteogenesis imperfecta (OI) is characterized by bone fragility and fractures that may be accompanied by bone deformity, dentinogenesis imperfecta, short stature, and shortened life span. About 90% of inpiduals with OI have dominant mutations in the type I collagen genesCOL1A1 and COL1A2. Recessive forms of OI resulting from mutations in collagen-modifying enzymes and chaperones CRTAP, LEPRE1, PPIB, and FKBP10 have recently been identified. We have identified an autosomal-recessive missense mutation (c.233T>C p.Leu78Pro) inSERPINH1, which encodes the collagen chaperone-like protein HSP47, that leads to a severe OI phenotype. The mutation results in degradation of the endoplasmic reticulum resident HSP47 via the proteasome. Type I procollagen accumulates in the Golgi of fibroblasts from the affected inpidual and a population of the secreted type I procollagen is protease sensitive. These findings suggest that HSP47 monitors the integrity of the triple helix of type I procollagen at the ER/cis-Golgi boundary and, when absent, the rate of transit from the ER to the Golgi is increased and helical structure is compromised. The normal 3-hydroxylation of the prolyl residue at position 986 of the triple helical domain of proa1(I) chains places the role of HSP47 downstream from the CRTAP/P3H1/CyPB complex that is involved in prolyl 3-hydroxylation. Identification of this mutation in SERPINH1 gives further insight into critical steps of the collagen biosynthetic pathway and the molecular pathogenesis of OI.

Identification of gene mutations by clinical laboratory testing in the Collagen Diagnostic Lab provides a wealth of data regarding clinical consequence of gene variants identified in familial aortopathies.  The disorders that we study include Ehlers-Danlos Syndrome type IV (COL3A1 gene mutations), and TGFB-related aortopathies caused by a number of genes including TGFBR1, TGFBR2, TGFB2, SMAD3, ACTA2 and others.  Abstracts of recent publications from the lab faculty and staff follow:

Survival is affected by mutation type and molecular mechanism in vascular Ehlers-Danlos syndrome (EDS type IV). Pepin MG et al. Genet Med 2014 June 12.

Purpose: We sought to characterize the natural history of vascular Ehlers-Danlos syndrome in inpiduals with heterozygous COL3A1 mutations.Methods:We reviewed clinical records for details of vascular, bowel, and organ complications in 1,231 inpiduals (630 index cases and 601 relatives).Results:Missense and splice-site mutations accounted for more than 90% of the 572 alterations that we had identified in COL3A1. Median survival was 51 years but was influenced by gender (lower in men) and by the type of mutation.Conclusion:Although vascular Ehlers-Danlos syndrome appears to be genetically homogeneous, allelic heterogeneity is marked, and the natural history varies with gender and type of mutation in COL3A1. These findings indicate that when counseling families, confirmation of the presence of a COL3A1 mutation and its nature can help evaluate the risks of complications. These data are also important ingredients in both the selection and allocation of inpiduals to appropriate arms in clinical trials to assess the effects of interventions.

Pregnancy-related deaths and complications in women with vascular Ehlers-Danlos syndrome. Murray ML et al. Genet Med 2014 June 12.

Purpose: The purpose of this study was to characterize the nature and magnitude of pregnancy risks in women with vascular Ehlers-Danlos syndrome.Methods:Pregnancy-related death rate was determined by a review of pedigrees of families with vascular Ehlers-Danlos syndrome. Maternal morbidity was characterized through semistructured interviews with women with vascular Ehlers-Danlos syndrome or their next of kin.Results:Pregnancy-related deaths occurred in 30 of 565 deliveries (5.3%). There was no difference in Kaplan-Meier survival curves between parous versus nulliparous women with vascular Ehlers-Danlos syndrome. Interviews with 39 women indicated that 46% of deliveries were uncomplicated. The most common pregnancy-related complications were third-/fourth-degree lacerations (20%) and preterm delivery (19%). Life-threatening complications occurred in 14.5% of deliveries and included arterial dissection/rupture (9.2%), uterine rupture (2.6%), and surgical complications (2.6%). There were 5 maternal deaths in 76 deliveries (6.5%).Conclusion:The risk of pregnancy-related complications is increased in women with vascular Ehlers-Danlos syndrome compared with the general population; however, survival data indicate that pregnancy does not appear to affect overall mortality compared with nulliparous women with vascular Ehlers-Danlos syndrome. The data were insufficient to determine whether mode or timing of delivery influenced risk of complications. Women with vascular Ehlers-Danlos syndrome should be engaged in a shared decision-making process when contemplating pregnancy and pregnancy management.

EDS type IV "null" nonsense mutations and missense mutations - comparison of survival and complications  Leistritz, DF et al. Genet Med 2011 Aug; 13(8):717-22.

Ehlers-Danlos syndrome (EDS) type IV, the vascular type, results from mutations in COL3A1, the gene that encodes the proα1(III) chain of type III procollagen. We have identified heterozygous COL3A1 mutations in 508 families, about 95% of which lead to the synthesis of an abnormal type III procollagen. Mutations that result in substitutions for glycine residues in the triple helical domain of the proα1(III) chain account for two-thirds of identified mutations in COL3A1, and splicing mutations comprise most of the remainder. We identified frameshift or premature termination codons that result in nonsense mediated mRNA decay of the COL3A1 mRNA encoded by that allele ("null mutations") in 19 families (about 4% of the total); cultured cells from these inpiduals produced about half the normal amount of type III procollagen and no abnormal molecules, as expected. We reviewed the clinical and family histories and medical complications in 53 inpiduals with COL3A1 null mutations. Compared to inpiduals with missense or exon-skipping mutations, we found that in the cohort withCOL3A1 null mutations mean life span was extended by close to 20 years, the age of first complication was delayed by almost 15 years, and major complications were limited to vascular events. The families were ascertained following a complication in a single inpidual but only 25% of relatives, some of whom had reached their 70s or 80s without incidents, had a complication and only 30% had any minor clinical features of EDS type IV. In families with osteogenesis imperfecta that results from mutations in the COL1A1 gene, more than 50% of affected inpiduals have haploinsufficiency mutations and have OI type I, the mildest OI phenotype. Because null mutations in COL3A1 are expected to be as common as those in COL1A1, our data suggest that they are far less penetrant than missense and splicing mutations yet may be causes of late onset arterial aneurysms. Since ascertainment on the basis of clinical findings alone has a low yield, genetic testing for COL3A1 mutations should be offered to all first degree relatives of an inpidual identified with a COL3A1 null mutation, and should be considered in those with compatible vascular complications at a young age and, potentially, in inpiduals with late onset arterial events.