Collagen XVIII Mutation in Knobloch Syndrome with Acute Lymphoblastic Leukemia

Abstract

Knobloch syndrome (KNO) is caused by mutations in the collagen XIII gene (COL18A1) and patients develop encephalocele and vitreoretinal degeneration. Here we report an El Salvadorian family where two sisters showed features of KNO. One of the siblings also developed acute lymphoblastic leukemia. DNA sequencing of COL18A1revealed a homozygous, 2-base pair deletion (c3514-3515delCT) in exon 41, which leads to abnormal collagen XVIII and deficiency of its proteolytic cleavage product endostatin. KNO patients with mutations in COL18A1 may be at risk for endostatin-related conditions including malignancy.

INTRODUCTION

Knobloch syndrome (KNO) is characterized by occipital scalp defects or encephalocele and ophthalmic findings that include high myopia, vitreoretinal degeneration, and retinal detachment.(Capstick 1975; Knobloch and Layer 1971; Seaver, et al. 1993; Sniderman, et al. 2000) Intellect is typically normal, with only a few reports of CNS anomalies and developmental delay. Knobloch syndrome is caused by DNA mutations in the collagen 18A gene (COL18A1, KNO1, chromosome 21q22.3), (Kliemann, et al. 2003; Sertie, et al. 1996) and has been found in families from Brazil, North America, Europe, India, and Algeria (Paisan-Ruiz, et al. 2009; Passos-Bueno, et al. 1994). Two other loci, KNO2 (unlinked) and KNO3 (linkage to chromosome 17q11.2) have also been implicated (Khaliq, et al. 2007; Menzel, et al. 2004; Wilson, et al. 1998).

COL18A1 codes for type XVIII collagen, a proteoglycan component of basement membranes including those of the iris, vitreous, and retina. In the eye, collagen XVIII plays a key role in ocular development, maintenance of visual function, and neuronal migration. Collagen XVIII is also cleaved near its C-terminus to produce endostatin, a potent inhibitor of angiogenesis and tumor growth (Digtyar, et al. 2007; Menzel, et al. 2004; Sertie, et al. 2000). Angiogenesis may be important in the pathogenesis of hematological cancer, and Collagen XVIII is associated with malignancy [(Feldman, et al. 2002; Hoffmann, et al. 2008; Iizasa, et al. 2004a; Iizasa, et al. 2004b; Lurje, et al. 2010; Ni, et al. 2009; Ohlund, et al. 2008; Ren, et al. 2003; Suzuki, et al. 2002a; Woo, et al. 2006)].

Over 48 patients in 16 families have been reported with this autosomal recessive condition, but there are no reports of patients with both Knobloch syndrome and malignancy. In this study, we describe a KNO1 family with a unique genotype where one affected sibling developed acute lymphoblastic leukemia (ALL).

METHODS

The study protocol was approved by the Institutional Review Board for Human Subjects Research (IRB) at the University of Iowa, was HIPPA compliant, and adhered to the tenets of the Declaration of Helsinki. The IRB waived informed consent for this retrospective chart study. Standard ophthalmological examinations were performed and fundus images were captured with the RetCam imaging system (Clarity Medical Systems, CA). Gene sequencing involved the exons and flanking 100bp intronic or 200bp promoter regions of the proband DNA ampliWed using the Platinum Pfx PCR kit (Invitrogen). Primer sequences are available upon request. PuriWed PCR ampliWcation products were electrophoresed on ABI 3100 and 3730 DNA sequencers by the Genomics Core.

RESULTS

The pedigree of an El Salvadorian family was represented by two parents (I-1, I-2) without known consanguinity, two daughters, and one son (Figure 1). The proband (II-3) was a 4-year-old female reported to have midline occipital soft tissue swelling and alopecia, hypotonia, mild developmental delay, and normal growth and OFC. A Brain MRI at 13 months of age showed an occipital dermal sinus tract, frontal cortical dysplasia, white matter loss, and mild ventriculomegaly. She had mild developmental delay. The proband’s 7 year old sister (II-2) had excision of a small occipital encephalocele at 1 month of age. She also had an occipital scalp defect, but normal growth and development. Brain MRI at age 4 showed an occipital dermal sinus tract and multiple punctuate areas of high signal along the supratentorial sulci. She was then diagnosed with acute lymphoblastic leukemia (ALL), pre-B type. Following treatment using the “AALL0031” regimen(Schultz, et al. 2009)], she successfully went into remission, was on maintenance, and completed her treatment at age 6-years. She remains in remission. The 11-year-old brother (II-1) was healthy. The proband and her sister had infantile nystagmus, high myopia, and poor vision, and they were referred for ophthalmic evaluation.

Figure 1.

Ophthalmic Findings

In the proband (II-3), the visual acuity measured 5/80 (20/320) OD and 5/63 (20/250). Cycloplegic refraction was −15.25 diopters OD and −14.75 0.50 090 diopters OS. A-scan echography confirmed high myopia by measuring axial eye lengths of 27.02 mm OD and 27.08 mm OS. The anterior segment exam was unremarkable. The intraocular pressures were 16 mmHg OU. A dilated fundus exam revealed vitreous attachment at the disc, syneresis, and small round condensations in sheets of anterior vitreous (Figure 2). These may represent persistent fetal hyaloid vasculature that has failed to regress completely (Duh, et al. 2004; Fukai, et al. 2002). The optic discs were small with peripapillary atrophy. There were visibly prominent choroidal vessels in the posterior pole and atrophic changes in the central macula. These white areas in the macula have been described as having the appearance of a macular coloboma. Examination of the peripheral retinas showed 360 degrees of irregular white dots and lines at the vitreoretinal interface. There were a few vitreoretinal adhesions temporally, but no lattice, retinal tears or detachment were found. There was a single sclerotic vessel in the right eye but no neovascularization.

Figure 2.

Similar exam findings were present in her older sister (II-2). The visual acuity measured 20/100 OD and 20/250 OS. Cycloplegic refraction was −18.00 diopters OU. A-scan echography confirmed high myopia by measuring axial eye lengths of 28.83 mm OD and 28.09 mm OS. There were vitreous sheets anteriorly with small round condensations and syneresis with attachment at the disc (Figure 3). There was an opacity on the left posterior lens capsule. The intraocular pressures were 11 mmHg OU. The posterior poles had staphylomatous changes with chorioretinal atrophy involving the fovea OD and a dense area of atrophy involving the fovea OS. In the peripheral retina there were the previously described irregular white dots and lines at the vitreoretinal interface. There were a few vitreoretinal adhesions temporally, but no lattice, retinal tears or detachment.

Figure 3.

Mutation Analysis

The combination of ophthalmic findings and encephaloceles in the daughters suggested Knobloch syndrome. The COL18A1 gene was sequenced in the two affected subjects and parents. A homozygous, 2-base pair deletion (c3514-3515delCT) was detected in exon 41 of both daughters (Figure 4). The identical heterozygous mutation was identified in each of the parents. This creates a frameshift at near amino acid 167 of the collagen XVIII protein, which is upstream from the proteolytic cleavage site of endostatin (O’Reilly, et al. 1997)

Figure 4.

DISCUSSION

This is the first Knobloch family originating from El Salvador, and the mutation represents a unique homozygous deletion on the COL18A1that was also recently reported in a family from India.(Paisan-Ruiz, et al. 2009) Affected individuals in the Indian family presented with predominantly neurological symptoms, such as ataxia, epilepsy, cognitive decline, and no leukemia, which emphasizes the emerging variability in the Knobloch disease phenotype. Although there was no known consanguinity, both parents were from the same region and there may be a founder effect. This mutation was previously reported in several unrelated KNO families. In these cases, however, affected subjects were compound heterozygotes with a different COL18A1 mutant companion.(Suzuki, et al. 2002a)None of the affected in these subjects developed ALL.

The homozygous mutation we describe causes a frameshift mutation that would eliminate the downstream proteolytic cleavage site for endostatin, suggesting that these patients may have no functional endostatin (Menzel, et al. 2004; Sertie, et al. 2000; Suzuki, et al. 2002b). Collagen XVIII is a structural protein, and might explain the connective tissue abnormalities of scalp defects and vitreoretinal degeneration found in Knobloch syndrome. The antiangiogenic properties of endostatin may be relevant to eye development, where persistent fetal vasculature has been reported in knockout mice, in a previously described family human family, and is suggested in images we present. (Duh, et al. 2004) The systemic effect of endostatin deficiency in Knobloch syndrome, however, is not known. (Digtyar, et al. 2007)

Based on the function of endostatin, it is interesting to speculate whether abnormal endostatin production might have contributed to the development of ALL in this family. Although ALL has not been previously reported, mutations in the terminal portion of the COL18A1have raised the possibility of defective enodstatin in KNO patients. Abnormal endostatin levels are found in a variety of human cancers (Digtyar, et al. 2007) and the antiangiogenic properties of endostatin may be therapeutic in some cases (Feldman, et al. 2002; Hoffmann, et al. 2008; Iizasa, et al. 2004a; Iizasa, et al. 2004b; Lurje, et al. 2010; Ni, et al. 2009; Ohlund, et al. 2008; Ren, et al. 2003; Schuch, et al. 2005; Suzuki, et al. 2002a; Woo, et al. 2006). Elevated plasma endostatin levels in some children with ALL have been detected, however, the mechanisms regulating angiogenesis and the role of endostatin in childhood ALL are poorly understood (Pui 1998; Schneider, et al. 2007). We recommend all Knobloch syndrome patients, particularly those with COL18A1 mutations affecting endostatin, have careful monitoring for the development of leukemia and other cancers.