RESEARCH ARTICLE
Autosomal Recessive Stickler Syndrome Due to a Loss of Function Mutation in the COL9A3 Gene Flavio Faletra,1* Adamo P. D’Adamo,2 Irene Bruno,1 Emmanouil Athanasakis,1 Saskia Biskup,3 Laura Esposito,4 and Paolo Gasparini1,2 1
Institute for Maternal and Child Health—IRCCS “Burlo Garofolo”—Trieste, Italy
2
University of Trieste, Trieste, Italy CeGaT GmbH, Tu¨bingen, Germany
3 4
CBM S.c.r.l. Area Science Park—Basovizza, Trieste, Italy
Manuscript Received: 7 March 2013; Manuscript Accepted: 8 July 2013
Stickler syndrome (STL) is a clinically variable and genetically heterogeneous syndrome characterized by ophthalmic, articular, orofacial, and auditory manifestations. STL has been described with both autosomal dominant and recessive inheritance. The dominant form is caused by mutations of COL2A1 (STL 1, OMIM 108300), COL11A1 (STL 2, OMIM 604841), and COL11A2 (STL 3, OMIM 184840) genes, while recessive forms have been associated with mutations of COL9A1 (OMIM 120210) and COL9A2 (OMIM 120260) genes. Type IX collagen is a heterotrimeric molecule formed by three genetically distinct chains: a1, a2, and a3 encoded by the COL9A1, COL9A2, and COL9A3 genes. Up to this time, only heterozygous mutations of COL9A3 gene have been reported in human and related to: (1) multiple epiphyseal dysplasia type 3, (2) susceptibility to an intervertebral disc disease, and (3) hearing loss. Here, we describe the first autosomal recessive Stickler family due to loss of function mutations (c.1176_1198del, p.Gln393Cysfs 25) of COL9A3 gene. These findings extend further the role of collagen genes family in the disease pathogenesis. Ó 2013 Wiley Periodicals, Inc.
Key words: COL9A3; stickler syndrome; autosomal recessive; c.1176_1198del; homozygous mutation
INTRODUCTION Stickler (STL) syndrome is a clinically variable and genetically heterogeneous syndrome, first described in 1965 [Stickler et al., 1965], and characterized by ophthalmic, articular, orofacial, and auditory involvement. Its incidence ranges approximately from 1 in 7.500 to 1 in 9.000 neonates. STL syndrome presents with a large clinical variability [Zlotogora et al., 1992] without any particular genotype–phenotype correlation. While the dominant forms are well known, only few cases of recessive forms have been reported. Type IX collagen is a structural component of hyaline cartilage, vitreous of the eye and intervertebral disc. It is a heterotrimeric molecule composed of three genetically distinct polypeptide chains: a1, a2, and a3 encoded by the COL9A1, COL9A2, and COL9A3
Ó 2013 Wiley Periodicals, Inc.
How to Cite this Article: Faletra F, D’Adamo AP, Bruno I, Athanasakis E, Biskup S, Esposito L, Gasparini P. 2014. Autosomal recessive stickler syndrome due to a loss of function mutation in the COL9A3 gene. Am J Med Genet Part A 164A:42–47.
genes. Proteins are characterized by three collagenous (COL1, COL2, and COL3, numbered from the C terminus), which are joined by four small noncollagenous domains (NC1–NC4). Mutations of COL9A1 gene can cause the multiple epiphyseal dysplasia (MED) 6 [Czarny-Ratajczak et al., 2001] (MED 6, OMIM 614135), and a recessive form of Stickler syndrome (STL) 4 [Van Camp et al., 2006] (STL 4, OMIM 614134), characterized by a severe sensorineural hearing loss, a moderate to high myopia with vitreoretinopathy, and an epiphyseal dysplasia. In 2006, Van Camp et al. reported a family showing the classical Stickler features including ocular, audiological and articular alterations [Van Camp et al., 2006]. Based on structural association between collagen types II and XI, the authors sequenced the COL9A1 gene, demonstrating the pathogenic homozygous p.Arg295 mutation. More recently, Nikopoulos et al. described two families with recessive COL9A1 mutations, one of them with the previously published p.Arg295 and the other with the novel p.Arg570 [Nikopoulos et al., 2011]. Instead, mutations of COL9A2 have been described in patients with MED 2 [Muragaki et al., 1996] (OMIM 600204) and in patients with an autosomal recessive STL 5 [Baker et al., 2011] (OMIM 614284).
Correspondence to: Flavio Faletra, Medical Genetics, Institute for Maternal and Child Health —IRCCS “Burlo Garofolo,” Via dell’Istria 65/1, Trieste, Italy. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 22 November 2013 DOI 10.1002/ajmg.a.36165
42
FALETRA ET AL. Moreover, mutated alleles of COL9A2 have been found with a high rate of development lumbar disc disease, caused by degeneration of intervertebral discs of the lumbar spine [Paassilta et al., 2001] (OMIM 603932). More recently, Baker et al. [2011] described a family with an autosomal recessive pattern of inheritance and a homozygous p.Asp281Glnfs 70 mutation in COL9A2 gene. Until now, there have been the following reports in reference to the COL9A3 gene: (A) heterozygous point mutations, causing (1) MED 3 [Lohiniva et al., 2000], sometimes associated with osteochondritis dissecans and a mild myopathy [Jackson et al., 2010]; (2) susceptibility to an intervertebral disc disease [Paassilta et al., 2001]; (3) hearing loss [Asamura et al., 2005]; and (B) large deletions causing complex phenotype including stiffness and pain with limited extension of joints in childhood, with some required surgery in adulthood [Traylor et al., 2010], but not MED. Herein, we report an intriguing case of a family (Figs. 1 and 2A) carrying an autosomal recessive loss of functions COL9A3 mutation and a STL syndrome with intellectual disability (ID).
MATERIALS AND METHODS Clinical Examination Three Moroccan siblings born from first-cousins healthy parents were referred for a genetic counseling because of hearing loss, visual
43 defects, bone abnormalities and ID. All the five family members (Fig. 1) were checked through general examination by otorhinolaryngologists, ophthalmologists, radiologists, child neuropsychiatrists, and geneticists. Serum parameters were also analyzed, including levels of alkaline phosphatase, parathyroid hormone, calcium, phosphorus, creatinine, urea, magnesium, and thyroid hormones. Hearing was evaluated by pure-tone audiometry: air and bone thresholds were determined at frequencies of 250, 500, 1,000, 2,000, 4,000, and 8,000 Hz, with intensities up to 120 dB. The puretone average (PTA) was calculated for all family members, using the average hearing thresholds at 500, 1,000, and 2,000 Hz.
Genetic Analysis Blood samples for DNA extraction were collected from all five family members with informed consent. DNA was extracted from leukocytes according to standard methods. In order to identify genes related to the phenotype we performed a homozygosity mapping using HumanCytoSNP-12 DNA Analysis BeadChip (Illumina, CA). We analyzed the obtained data with Plink [Purcell et al., 2007] software using the following parameters: length of the sliding window ¼ 1,000 kb; number of SNPs in a windows ¼ 100 and a minimum density of 1 SNP/kb. Primers were designed for the amplification of the coding region and exon–
FIG. 1. Phenotypic features of the three affected family members and their parents.
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AMERICAN JOURNAL OF MEDICAL GENETICS PART A
FIG. 2. (A) Siblings Genealogical tree of the family carrying the COL9A3 mutation; (B) audiograms of the three affected siblings. No difference has been shown between the bone and the air conduction; (C) partial electropherogram showing the homozygous c.1176_1198del mutation (NP_001844.3: p.Gln393Cysfs 25).
intron boundaries of the COL9A3, NEUROD2, NLGN1, and SLC4A10 genes. We performed direct sequencing of the PCR products by standard procedures using an ABI PRISM 3110 Genetic Analyzer (Life Technologies, CA). All the new variants were analyzed with the online predictors of pathogenicity PolyPhen2 [Adzhubei et al., 2010] and MutationTaster [Schwarz et al., 2010]. A hundred healthy matched controls were genotyped for the new variants.
RESULTS Clinical Findings No alterations in blood parameters have been detected. Height/ weight values were between the 50th and 75th centile, 107 cm/16 kg, 144 cm/38 kg, and 170 cm/60 kg for the three children respectively (age/sex 4/f, 11/m, 16/m). The two younger siblings have a slight flat profile with midface hypoplasia and depressed nasal bridge. Additional features include downslanted palpebral fissures in the two boys and a ptosis in the older one (Fig. 1). The three affected family members showed a moderate-to high myopia (6, 8, and 5), astigmatism and amblyopia due to impairment of ocular motility. No vitreous or retinal abnormalities have been detected, despite the oldest brother showed an ERG at the lower limits of the normal range. We found a symmetric, sensorineural and moderate to severe hearing loss, which was treated using hearing aids. The audiograms (Fig. 2B) showed a mildly down-sloping form with an age-dependent correlation. Conversely, the parents of the three children did not have any hearing loss. The PTAs were 34.6, 50.0, and 59.3 for the probands. We noted an internal rotation of the tibia, surgically corrected, a pes planus with a variable degree, and non-specific metacarpal and femoral alteration in epiphysis (Fig. 3). No vertebral alterations were detected. Finally, the three siblings showed a moderate to severe ID.
Genetic Analysis The nine homozygous regions shared by all the three affected siblings (Supplemental eTable S1 in supporting information online) have been analyzed in order to identify the genes potentially causing hearing loss, ocular, bone alterations, and ID. Since no genes related to the complete clinical picture were found, the ID was separated from the rest of the phenotype. For the physical features the selected and sequenced COL9A3 gene (OMIM 120270) revealed a homozygous frameshift mutation in the three siblings: c.1176_1198del (NP_001844.3: p. Gln393Cysfs 25; Fig. 2C). This mutation affects the exon 23, which encodes for part of the COL2 domain of COL9A3 and leads to a premature stop codon. Both parents were heterozygous for the mutation. The mutation was not reported or listed in dbSNP (www.ncbi.nlm.nih.gov/projects/SNP/Build 137) or NHLBI-ESP (http://evs.gs.washigton.edu/EVS/) databases and none of the 100 matched healthy controls showed the same mutation. For the ID we selected and sequenced three genes based to their functions: NEUROD2 (OMIM 601725) involved a neuronal differentiation [Sugimoto et al., 2009] and mimicking a cretinism in neuroD2-deficient mice [Lin et al., 2006]; NLGN1 (OMIM 600568), involved a formation of synaptic contacts [Reissner et al., 2008] and it is an important candidate gene for autism [Ylisaukko-oja et al., 2005]; SLC4A10 (OMIM 605556), an Na(þ)dependent Cl/HCO3 exchanger widely expressed in the central nervous system [Jacobs et al., 2008], similar to SLC4A4 (OMIM 603345) gene, responsible for a renal tubular acidosis with ID and found interrupted by a deletion in a girl with ID [Gurnett et al., 2008]. We did not detect pathological copy number variations or mutations in NEUROD2, NLGN1, and SLC4A10 genes in the three affected siblings.
FALETRA ET AL.
45
FIG. 3. Slight bone abnormalities shared by the three affected children: (A) hand of II.1 individual with squared metacarpal epiphysis; (B) hips and legs of II.3 with squared femoral epiphysis and internal tibial rotation; (C) foot of II.2 severe bilateral flat feet with valgus hindfoot.
DISCUSSION Recently COL9A1 and COL9A2 homozygous mutations have been described in four families [Van Camp et al., 2006; Baker et al., 2011; Nikopoulos et al., 2011]. Clinical features reported in these patients, as well as those of dominant STL and Marshall syndromes, are overlapping to what we found in our family (Table I). Classical signs of STL syndrome affect: (a) eyes, with myopia, vitreoretinal degeneration, cataracts, and retinal detachment. STL 3 has no ocular
findings because in this organ COL11A2 is replaced by COL5A2 [Mayne et al., 1993]; (b) ears, with a sensorineural, mixed, and/or conductive hearing loss; (c) joints, with hypermobility, mild spondyloepiphyseal dysplasia, and precocious osteoarthritis; (d) craniofacial structures, causing abnormalities as midface hypoplasia, depressed nasal bridge, anteverted nares, bifid uvula, cleft hard palate, micrognathia, and Pierre Robin sequence. As a matter of fact, patients with STL syndrome share a series of features including hearing loss, phenotypic and ocular features as those
TABLE I. Clinical Finding in Autosomal Dominant and Recessive Stickler Syndrome Clinical findings Clinical findings in stickler syndrome High myopia Vitreoretinal degeneration Retinal detachment Cataracts Hearing loss Mid-face hypoplasia Cleft palate/Pierre Robin sequence Anteverted nares Small chin Short stature Spondyloepiphyseal dysplasia Early-onset osteoarthritis Other findings
“”, absent; “þ”, present; “?”, not known.
STL1 and STL2 (COL2A1 and COL11A1)
STL3 (COL11A2)
COL9A1 Arg295
COL9A2 Asp281Glnfs 70
COL9A3 Gln393Cysfs 25
þ þ þ þ þ þ þ þ þ þ/ þ þ
þ þ þ þ þ þ/ þ þ
þ þ þ þ þ ? Genua valga
þ þ þ þ þ þ þ ? ?
þ þ þ Internal tibial rotation, pes planus, downslanted palpebral fissures
46 characterizing our patients. Conversely, the mild and proportionate short stature reported in recessive COL9A1 and COL9A2 mutations is not present in our probands, showing a height and weight ranging between the 50th and the 75th centiles. Collagen IX is a member of fibril-associated collagens with interrupted triple helices (FACIT), which do not form fibrils themselves but are attached to the surfaces of pre-existing fibrils [Myllyharju and Kivirikko, 2001]. Indeed, its fibrils are regularly aligned along the fibrils of collagen II and XI. Moreover, the deficiency of a1 (IX) chains in mice leads to a functional knockout of all the collagen IX, even though other chains were normally transcribed [Hagg et al., 1997]. These results suggest that the loss of function of any of the collagen IX chains leads to a loss of function of collagen IX. Therefore, the homozygous c.1176_1198 deletion found in the affected family members leads to a premature stop codon causing a loss of function of the entire collagen IX. Until now, heterozygous mutations in all three COL9 genes have been reported causing different autosomal dominant MED. These are related to hearing loss (only COL9A3) and to the increasing of the susceptibility of an intervertebral disc disease (COL9A2 and COL9A3). Recessive mutations in COL9 have been described only for the chains a1 [Van Camp et al., 2006] and a2 [Baker et al., 2011] in humans, and have been related to a recessive form of STL. Despite this, Goldstein et al. studied an oculo-skeletal dysplasia (OSD) [Goldstein et al., 2010] and identified two different causative loci in Labrador Retriever and Samoyed dog breeds, one of which was found to co-segregate with a one-base deletion in exon 1 of COL9A3 gene. This means that the relationship between loss of function mutations of COL9A3 gene and a canine autosomal recessive form of OSD, close to human hereditary arthro-ophtalmopathies such as Stickler or Marshall syndromes, has been previously hypothesized. We note several important points: (1) despite STL syndrome having a large clinical variability [Zlotogora et al., 1992] clinical features of our patients overlap with those previously reported; (2) loss of function of any of the collagen IX chain leads to a loss of function of the entire collagen IX; (3) recessive mutations of COL9A1 and COL9A2 have been already associated with STL syndrome; (4) an arthro-ophtalmopathies due to the recessive mutation of COL9A3 has been already described in dogs; and (5) the healthy status of the heterozygous carriers of COL9A3 p. Gln393Cysfs 25 in our family. Due to these five points we suggest a close relation between recessive loss of function mutations of COL9A3 and STL syndrome. Conversely, on one hand the ID in the three siblings seems not to be explainable by the alteration of the collagen IX and on the other hand we did not detect any mutations of NEUROD2, NLGN1, SLC4A10 genes. Obviously, several other genes located in the shared homozygous regions with unclear or unknown functions could be causative. In conclusion, COL9A3 is likely to be a new gene that can cause an autosomal recessive STL. Mutation analysis of this gene is recommended for patients with STL and autosomal recessive inheritance. Obviously, due to the absence of ID in all probands with autosomal dominant or recessive STL, at this point we cannot hypothesize a pathogenetic role of COL9A3 for this manifestation. Further families will have to be studied to perform genotype–phenotype correlations.
AMERICAN JOURNAL OF MEDICAL GENETICS PART A
ACKNOWLEDGMENTS The authors would like to thank the family who participated in this study and Flora Maria Murru and Floriana Zennaro for their technical support in the radiograms’ interpretation.
REFERENCES Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR. 2010. A method and server for predicting damaging missense mutations. Nat Methods 7:248–249. Asamura K, Abe S, Fukuoka H, Nakamura Y, Usami S. 2005. Mutation analysis of COL9A3, a gene highly expressed in the cochlea, in hearing loss patients. Auris Nasus Larynx 32:113–117. Baker S, Booth C, Fillman C, Shapiro M, Blair MP, Hyland JC, Ala-Kokko L. 2011. A loss of function mutation in the COL9A2 gene causes autosomal recessive Stickler syndrome. Am J Med Genet A 155A:1668–1672. Czarny-Ratajczak M, Lohiniva J, Rogala P, Kozlowski K, Pera¨la¨ M, arter L, Spector TD, Kolodziej L, Seppa¨nen U, Glazar R, Kro´lewski J, LatosBielenska A, Ala-Kokko L. 2001. A mutation in COL9A1 causes multiple epiphyseal dysplasia: Further evidence for locus heterogeneity. Am J Hum Genet 69:969–980. Goldstein O, Guyon R, Kukekova A, Kuznetsova TN, Pearce-Kelling SE, Johnson J, Aguirre GD, Acland GM. 2010. COL9A2 and COL9A3 mutations in canine autosomal recessive oculoskeletal dysplasia. Mamm Genome 21:398–408. Gurnett CA, Veile R, Zempel J, Blackburn L, Lovett M, Bowcock A. 2008. Disruption of sodium bicarbonate transporter SLC4A10 in a patient with complex partial epilepsy and mental retardation. Arch Neurol 65:550–553. Hagg R, Hedbom E, Mo¨llers U, Aszo´di A, Fa¨ssler R, Bruckner P. 1997. Absence of the alpha1(IX) chain leads to a functional knock-out of the entire collagen IX protein in mice. J Biol Chem 272:20650–20654. Jackson GC, Marcus-Soekarman D, Stolte-Dijkstra I, Verrips A, Taylor JA, Briggs MD. 2010. Type IX collagen gene mutations can result in multiple epiphyseal dysplasia that is associated with osteochondritis dissecans and a mild myopathy. Am J Med Genet A 152A:863–869. Jacobs S, Ruusuvuori E, Sipila¨ ST, Haapanen A, Damkier HH, Kurth I, Hentschke M, Schweizer M, Rudhard Y, Laatikainen LM, Tyynela¨ J, Praetorius J, Voipio J, Hu¨bner CA. 2008. Mice with targeted Slc4a10 gene disruption have small brain ventricles and show reduced neuronal excitability. Proc Natl Acad Sci USA 105:311–316. Lin CH, Tapscott SJ, Olson JM. 2006. Congenital hypothyroidism (cretinism) in neuroD2-deficient mice. Mol Cell Biol 26:4311–4315. Lohiniva J, Paassilta P, Seppa¨nen U, Vierimaa O, Kivirikko S, Ala-Kokko L. 2000. Splicing mutations in the COL3 domain of collagen IX cause multiple epiphyseal dysplasia. Am J Med Genet 90:216–222. Mayne R, Brewton RG, Mayne PM, Baker JR. 1993. Isolation and characterization of the chains of type V/type XI collagen present in bovine vitreous. J Biol Chem 268:9381–9386. Muragaki Y, Mariman EC, van Beersum SE, Pera¨la¨ M, van Mourik JB, Warman ML, Olsen BR, Hamel BC. 1996. A mutation in the gene encoding the alpha 2 chain of the fibril-associated collagenIX, COL9A2, causes multiple epiphyseal dysplasia (EDM2). Nat Genet 12:103–105. Myllyharju J, Kivirikko KI. 2001. Collagens and collagen-related diseases. Ann Med 33:7–21. Nikopoulos K, Schrauwen I, Simon M, Collin RW, Veckeneer M, Keymolen K, Van Camp G, Cremers FP, van den Born LI. 2011. Autosomal
FALETRA ET AL. recessive Stickler syndrome in two families is caused by mutations in the COL9A1 gene. Invest Ophthalmol Vis Sci 52:4774–4779. Paassilta P, Lohiniva J, Go¨ring HH, Pera¨la¨ M, Ra¨ina¨ SS, Karppinen J, Hakala M, Palm T, Kro¨ger H, Kaitila I, Vanharanta H, Ott J, Ala-Kokko L. 2001. Identification of a novel common genetic risk factor for lumbar disk disease. JAMA 285:1843–1849. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Maller J, Sklar P, de Bakker PI, Daly MJ, Sham PC. 2007. PLINK: A toolset for whole-genome association and population-based linkage analysis. Am J Hum Genet 81:559–575. Reissner C, Klose M, Fairless R, Missler M. 2008. Mutational analysis of the neurexin/neuroligin complex reveals essential and regulatory components. Proc Natl Acad Sci USA 105:15124–15129. Schwarz JM, Ro¨delsperger C, Schuelke M, Seelow D. 2010. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods 7:575–576.
47 Traylor RN, Bruno DL, Burgess T, Wildin R, Spencer A, Ganesamoorthy D, Amor DJ, Hunter M, Caplan M, Rosenfeld JA, Theisen A, Torchia BS, Shaffer LG, Ballif BC, Slater HR. 2010. A genotype-first approach for the molecular and clinical characterization of uncommon de novo microdeletion of 20q13. PLoS ONE 5: e12462. Van Camp G, Snoeckx RL, Hilgert N, van den Ende J, Fukuoka H, Wagatsuma M, Suzuki H, Smets RM, Vanhoenacker F, Declau F, Van de Heyning P, Usami S. 2006. A new autosomal recessive form of Stickler syndrome is caused by a mutation in the COL9A1 gene. Am J Hum Genet 79:449–457. Ylisaukko-oja T, Rehnstro¨m K, Auranen M, Vanhala R, Alen R, Kempas E, Ellonen P, Turunen JA, Makkonen I, Riikonen R, Nieminen-von Wendt T, von Wendt L, Peltonen L, Ja¨rvela¨ I. 2005. Analysis of four neuroligin genes as candidates for autism. Eur J Hum Genet 13:1285–1292. Zlotogora J, Sagi M, Shuper A, Leiba H, Merin S. 1992. Variability of Stickler syndrome. Am J Med Genet 42:337–339.
Stickler GB, Belau PG, Farrell FJ, Jones JD, Jugh DG, Steinberg AG, Ward LE. 1965. Hereditary progressive arthro-ophthalmopathy. Mayo Clin Proc 40:433–455.
SUPPORTING INFORMATION
Sugimoto Y, Furuno T, Nakanishi M. 2009. Effect of NeuroD2 expression on neuronal differentiation in mouse embryonic stem cells. Cell Biol Int 33:174–179.
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Autosomal Recessive Stickler Syndrome Due to a Loss of Function Mutation in the COL9A3 Gene Flavio Faletra,1* Adamo P. D’Adamo,2 Irene Bruno,1 Emmanouil Athanasakis,1 Saskia Biskup,3 Laura Esposito,4 and Paolo Gasparini1,2 1
Institute for Maternal and Child Health—IRCCS “Burlo Garofolo”—Trieste, Italy
2
University of Trieste, Trieste, Italy CeGaT GmbH, Tu¨bingen, Germany
3 4
CBM S.c.r.l. Area Science Park—Basovizza, Trieste, Italy
Manuscript Received: 7 March 2013; Manuscript Accepted: 8 July 2013
Stickler syndrome (STL) is a clinically variable and genetically heterogeneous syndrome characterized by ophthalmic, articular, orofacial, and auditory manifestations. STL has been described with both autosomal dominant and recessive inheritance. The dominant form is caused by mutations of COL2A1 (STL 1, OMIM 108300), COL11A1 (STL 2, OMIM 604841), and COL11A2 (STL 3, OMIM 184840) genes, while recessive forms have been associated with mutations of COL9A1 (OMIM 120210) and COL9A2 (OMIM 120260) genes. Type IX collagen is a heterotrimeric molecule formed by three genetically distinct chains: a1, a2, and a3 encoded by the COL9A1, COL9A2, and COL9A3 genes. Up to this time, only heterozygous mutations of COL9A3 gene have been reported in human and related to: (1) multiple epiphyseal dysplasia type 3, (2) susceptibility to an intervertebral disc disease, and (3) hearing loss. Here, we describe the first autosomal recessive Stickler family due to loss of function mutations (c.1176_1198del, p.Gln393Cysfs 25) of COL9A3 gene. These findings extend further the role of collagen genes family in the disease pathogenesis. Ó 2013 Wiley Periodicals, Inc.
Key words: COL9A3; stickler syndrome; autosomal recessive; c.1176_1198del; homozygous mutation
INTRODUCTION Stickler (STL) syndrome is a clinically variable and genetically heterogeneous syndrome, first described in 1965 [Stickler et al., 1965], and characterized by ophthalmic, articular, orofacial, and auditory involvement. Its incidence ranges approximately from 1 in 7.500 to 1 in 9.000 neonates. STL syndrome presents with a large clinical variability [Zlotogora et al., 1992] without any particular genotype–phenotype correlation. While the dominant forms are well known, only few cases of recessive forms have been reported. Type IX collagen is a structural component of hyaline cartilage, vitreous of the eye and intervertebral disc. It is a heterotrimeric molecule composed of three genetically distinct polypeptide chains: a1, a2, and a3 encoded by the COL9A1, COL9A2, and COL9A3
Ó 2013 Wiley Periodicals, Inc.
How to Cite this Article: Faletra F, D’Adamo AP, Bruno I, Athanasakis E, Biskup S, Esposito L, Gasparini P. 2014. Autosomal recessive stickler syndrome due to a loss of function mutation in the COL9A3 gene. Am J Med Genet Part A 164A:42–47.
genes. Proteins are characterized by three collagenous (COL1, COL2, and COL3, numbered from the C terminus), which are joined by four small noncollagenous domains (NC1–NC4). Mutations of COL9A1 gene can cause the multiple epiphyseal dysplasia (MED) 6 [Czarny-Ratajczak et al., 2001] (MED 6, OMIM 614135), and a recessive form of Stickler syndrome (STL) 4 [Van Camp et al., 2006] (STL 4, OMIM 614134), characterized by a severe sensorineural hearing loss, a moderate to high myopia with vitreoretinopathy, and an epiphyseal dysplasia. In 2006, Van Camp et al. reported a family showing the classical Stickler features including ocular, audiological and articular alterations [Van Camp et al., 2006]. Based on structural association between collagen types II and XI, the authors sequenced the COL9A1 gene, demonstrating the pathogenic homozygous p.Arg295 mutation. More recently, Nikopoulos et al. described two families with recessive COL9A1 mutations, one of them with the previously published p.Arg295 and the other with the novel p.Arg570 [Nikopoulos et al., 2011]. Instead, mutations of COL9A2 have been described in patients with MED 2 [Muragaki et al., 1996] (OMIM 600204) and in patients with an autosomal recessive STL 5 [Baker et al., 2011] (OMIM 614284).
Correspondence to: Flavio Faletra, Medical Genetics, Institute for Maternal and Child Health —IRCCS “Burlo Garofolo,” Via dell’Istria 65/1, Trieste, Italy. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 22 November 2013 DOI 10.1002/ajmg.a.36165
42
FALETRA ET AL. Moreover, mutated alleles of COL9A2 have been found with a high rate of development lumbar disc disease, caused by degeneration of intervertebral discs of the lumbar spine [Paassilta et al., 2001] (OMIM 603932). More recently, Baker et al. [2011] described a family with an autosomal recessive pattern of inheritance and a homozygous p.Asp281Glnfs 70 mutation in COL9A2 gene. Until now, there have been the following reports in reference to the COL9A3 gene: (A) heterozygous point mutations, causing (1) MED 3 [Lohiniva et al., 2000], sometimes associated with osteochondritis dissecans and a mild myopathy [Jackson et al., 2010]; (2) susceptibility to an intervertebral disc disease [Paassilta et al., 2001]; (3) hearing loss [Asamura et al., 2005]; and (B) large deletions causing complex phenotype including stiffness and pain with limited extension of joints in childhood, with some required surgery in adulthood [Traylor et al., 2010], but not MED. Herein, we report an intriguing case of a family (Figs. 1 and 2A) carrying an autosomal recessive loss of functions COL9A3 mutation and a STL syndrome with intellectual disability (ID).
MATERIALS AND METHODS Clinical Examination Three Moroccan siblings born from first-cousins healthy parents were referred for a genetic counseling because of hearing loss, visual
43 defects, bone abnormalities and ID. All the five family members (Fig. 1) were checked through general examination by otorhinolaryngologists, ophthalmologists, radiologists, child neuropsychiatrists, and geneticists. Serum parameters were also analyzed, including levels of alkaline phosphatase, parathyroid hormone, calcium, phosphorus, creatinine, urea, magnesium, and thyroid hormones. Hearing was evaluated by pure-tone audiometry: air and bone thresholds were determined at frequencies of 250, 500, 1,000, 2,000, 4,000, and 8,000 Hz, with intensities up to 120 dB. The puretone average (PTA) was calculated for all family members, using the average hearing thresholds at 500, 1,000, and 2,000 Hz.
Genetic Analysis Blood samples for DNA extraction were collected from all five family members with informed consent. DNA was extracted from leukocytes according to standard methods. In order to identify genes related to the phenotype we performed a homozygosity mapping using HumanCytoSNP-12 DNA Analysis BeadChip (Illumina, CA). We analyzed the obtained data with Plink [Purcell et al., 2007] software using the following parameters: length of the sliding window ¼ 1,000 kb; number of SNPs in a windows ¼ 100 and a minimum density of 1 SNP/kb. Primers were designed for the amplification of the coding region and exon–
FIG. 1. Phenotypic features of the three affected family members and their parents.
44
AMERICAN JOURNAL OF MEDICAL GENETICS PART A
FIG. 2. (A) Siblings Genealogical tree of the family carrying the COL9A3 mutation; (B) audiograms of the three affected siblings. No difference has been shown between the bone and the air conduction; (C) partial electropherogram showing the homozygous c.1176_1198del mutation (NP_001844.3: p.Gln393Cysfs 25).
intron boundaries of the COL9A3, NEUROD2, NLGN1, and SLC4A10 genes. We performed direct sequencing of the PCR products by standard procedures using an ABI PRISM 3110 Genetic Analyzer (Life Technologies, CA). All the new variants were analyzed with the online predictors of pathogenicity PolyPhen2 [Adzhubei et al., 2010] and MutationTaster [Schwarz et al., 2010]. A hundred healthy matched controls were genotyped for the new variants.
RESULTS Clinical Findings No alterations in blood parameters have been detected. Height/ weight values were between the 50th and 75th centile, 107 cm/16 kg, 144 cm/38 kg, and 170 cm/60 kg for the three children respectively (age/sex 4/f, 11/m, 16/m). The two younger siblings have a slight flat profile with midface hypoplasia and depressed nasal bridge. Additional features include downslanted palpebral fissures in the two boys and a ptosis in the older one (Fig. 1). The three affected family members showed a moderate-to high myopia (6, 8, and 5), astigmatism and amblyopia due to impairment of ocular motility. No vitreous or retinal abnormalities have been detected, despite the oldest brother showed an ERG at the lower limits of the normal range. We found a symmetric, sensorineural and moderate to severe hearing loss, which was treated using hearing aids. The audiograms (Fig. 2B) showed a mildly down-sloping form with an age-dependent correlation. Conversely, the parents of the three children did not have any hearing loss. The PTAs were 34.6, 50.0, and 59.3 for the probands. We noted an internal rotation of the tibia, surgically corrected, a pes planus with a variable degree, and non-specific metacarpal and femoral alteration in epiphysis (Fig. 3). No vertebral alterations were detected. Finally, the three siblings showed a moderate to severe ID.
Genetic Analysis The nine homozygous regions shared by all the three affected siblings (Supplemental eTable S1 in supporting information online) have been analyzed in order to identify the genes potentially causing hearing loss, ocular, bone alterations, and ID. Since no genes related to the complete clinical picture were found, the ID was separated from the rest of the phenotype. For the physical features the selected and sequenced COL9A3 gene (OMIM 120270) revealed a homozygous frameshift mutation in the three siblings: c.1176_1198del (NP_001844.3: p. Gln393Cysfs 25; Fig. 2C). This mutation affects the exon 23, which encodes for part of the COL2 domain of COL9A3 and leads to a premature stop codon. Both parents were heterozygous for the mutation. The mutation was not reported or listed in dbSNP (www.ncbi.nlm.nih.gov/projects/SNP/Build 137) or NHLBI-ESP (http://evs.gs.washigton.edu/EVS/) databases and none of the 100 matched healthy controls showed the same mutation. For the ID we selected and sequenced three genes based to their functions: NEUROD2 (OMIM 601725) involved a neuronal differentiation [Sugimoto et al., 2009] and mimicking a cretinism in neuroD2-deficient mice [Lin et al., 2006]; NLGN1 (OMIM 600568), involved a formation of synaptic contacts [Reissner et al., 2008] and it is an important candidate gene for autism [Ylisaukko-oja et al., 2005]; SLC4A10 (OMIM 605556), an Na(þ)dependent Cl/HCO3 exchanger widely expressed in the central nervous system [Jacobs et al., 2008], similar to SLC4A4 (OMIM 603345) gene, responsible for a renal tubular acidosis with ID and found interrupted by a deletion in a girl with ID [Gurnett et al., 2008]. We did not detect pathological copy number variations or mutations in NEUROD2, NLGN1, and SLC4A10 genes in the three affected siblings.
FALETRA ET AL.
45
FIG. 3. Slight bone abnormalities shared by the three affected children: (A) hand of II.1 individual with squared metacarpal epiphysis; (B) hips and legs of II.3 with squared femoral epiphysis and internal tibial rotation; (C) foot of II.2 severe bilateral flat feet with valgus hindfoot.
DISCUSSION Recently COL9A1 and COL9A2 homozygous mutations have been described in four families [Van Camp et al., 2006; Baker et al., 2011; Nikopoulos et al., 2011]. Clinical features reported in these patients, as well as those of dominant STL and Marshall syndromes, are overlapping to what we found in our family (Table I). Classical signs of STL syndrome affect: (a) eyes, with myopia, vitreoretinal degeneration, cataracts, and retinal detachment. STL 3 has no ocular
findings because in this organ COL11A2 is replaced by COL5A2 [Mayne et al., 1993]; (b) ears, with a sensorineural, mixed, and/or conductive hearing loss; (c) joints, with hypermobility, mild spondyloepiphyseal dysplasia, and precocious osteoarthritis; (d) craniofacial structures, causing abnormalities as midface hypoplasia, depressed nasal bridge, anteverted nares, bifid uvula, cleft hard palate, micrognathia, and Pierre Robin sequence. As a matter of fact, patients with STL syndrome share a series of features including hearing loss, phenotypic and ocular features as those
TABLE I. Clinical Finding in Autosomal Dominant and Recessive Stickler Syndrome Clinical findings Clinical findings in stickler syndrome High myopia Vitreoretinal degeneration Retinal detachment Cataracts Hearing loss Mid-face hypoplasia Cleft palate/Pierre Robin sequence Anteverted nares Small chin Short stature Spondyloepiphyseal dysplasia Early-onset osteoarthritis Other findings
“”, absent; “þ”, present; “?”, not known.
STL1 and STL2 (COL2A1 and COL11A1)
STL3 (COL11A2)
COL9A1 Arg295
COL9A2 Asp281Glnfs 70
COL9A3 Gln393Cysfs 25
þ þ þ þ þ þ þ þ þ þ/ þ þ
þ þ þ þ þ þ/ þ þ
þ þ þ þ þ ? Genua valga
þ þ þ þ þ þ þ ? ?
þ þ þ Internal tibial rotation, pes planus, downslanted palpebral fissures
46 characterizing our patients. Conversely, the mild and proportionate short stature reported in recessive COL9A1 and COL9A2 mutations is not present in our probands, showing a height and weight ranging between the 50th and the 75th centiles. Collagen IX is a member of fibril-associated collagens with interrupted triple helices (FACIT), which do not form fibrils themselves but are attached to the surfaces of pre-existing fibrils [Myllyharju and Kivirikko, 2001]. Indeed, its fibrils are regularly aligned along the fibrils of collagen II and XI. Moreover, the deficiency of a1 (IX) chains in mice leads to a functional knockout of all the collagen IX, even though other chains were normally transcribed [Hagg et al., 1997]. These results suggest that the loss of function of any of the collagen IX chains leads to a loss of function of collagen IX. Therefore, the homozygous c.1176_1198 deletion found in the affected family members leads to a premature stop codon causing a loss of function of the entire collagen IX. Until now, heterozygous mutations in all three COL9 genes have been reported causing different autosomal dominant MED. These are related to hearing loss (only COL9A3) and to the increasing of the susceptibility of an intervertebral disc disease (COL9A2 and COL9A3). Recessive mutations in COL9 have been described only for the chains a1 [Van Camp et al., 2006] and a2 [Baker et al., 2011] in humans, and have been related to a recessive form of STL. Despite this, Goldstein et al. studied an oculo-skeletal dysplasia (OSD) [Goldstein et al., 2010] and identified two different causative loci in Labrador Retriever and Samoyed dog breeds, one of which was found to co-segregate with a one-base deletion in exon 1 of COL9A3 gene. This means that the relationship between loss of function mutations of COL9A3 gene and a canine autosomal recessive form of OSD, close to human hereditary arthro-ophtalmopathies such as Stickler or Marshall syndromes, has been previously hypothesized. We note several important points: (1) despite STL syndrome having a large clinical variability [Zlotogora et al., 1992] clinical features of our patients overlap with those previously reported; (2) loss of function of any of the collagen IX chain leads to a loss of function of the entire collagen IX; (3) recessive mutations of COL9A1 and COL9A2 have been already associated with STL syndrome; (4) an arthro-ophtalmopathies due to the recessive mutation of COL9A3 has been already described in dogs; and (5) the healthy status of the heterozygous carriers of COL9A3 p. Gln393Cysfs 25 in our family. Due to these five points we suggest a close relation between recessive loss of function mutations of COL9A3 and STL syndrome. Conversely, on one hand the ID in the three siblings seems not to be explainable by the alteration of the collagen IX and on the other hand we did not detect any mutations of NEUROD2, NLGN1, SLC4A10 genes. Obviously, several other genes located in the shared homozygous regions with unclear or unknown functions could be causative. In conclusion, COL9A3 is likely to be a new gene that can cause an autosomal recessive STL. Mutation analysis of this gene is recommended for patients with STL and autosomal recessive inheritance. Obviously, due to the absence of ID in all probands with autosomal dominant or recessive STL, at this point we cannot hypothesize a pathogenetic role of COL9A3 for this manifestation. Further families will have to be studied to perform genotype–phenotype correlations.
AMERICAN JOURNAL OF MEDICAL GENETICS PART A
ACKNOWLEDGMENTS The authors would like to thank the family who participated in this study and Flora Maria Murru and Floriana Zennaro for their technical support in the radiograms’ interpretation.
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SUPPORTING INFORMATION
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