BRIEF REPORT

Novel recessive BFSP2 and PITX3 mutations: Insights into mutational mechanisms from consanguineous populations Mohammed A. Aldahmesh, BSc, MSc, PhD1, Arif O. Khan, MD1,2, Jawahir Mohamed, BSc1, and Fowzan S. Alkuraya, MD1,3,4

Purpose: Designating mutations as recessive or dominant is a function of mutations often provide new insights into the molecular patho- 1 the effect of the mutant allele on the phenotype. in which both genesis of the mutation and the associated phenotype. classes of mutations are known to exist are particularly interesting to study We have shown that for genetically heterogeneous disorders because these mutations typically define distinct pathogenic mechanisms at that can be caused by both dominant and recessive mutations, the molecular level. Methods: We studied two consanguineous families recessive mutations are typically overrepresented in consan- 2 with different eye phenotypes and used a combination of candidate guineous populations. However, we share here an emerging analysis and homozygosity mapping to identify the underlying genetic theme wherein even for genes in which only dominant muta- defects. Results: In one family, a novel BFSP2 mutation causes autosomal tions are known to exist, consanguineous populations can reveal recessive diffuse cortical cataract with scattered lens opacities, and in the presence of recessively acting mutations which adds a new another, a novel PITX3 mutation causes an autosomal recessive severe dimension to the mechanistic characterization of the molecular form of anterior segment dysgenesis and . Conclusion: We phenotype associated with these genes. show that BFSP2 and PITX3, hitherto known to cause eye defects only in a dominant fashion, can also present recessively. The likely null nature of MATERIALS AND METHODS both mutations and lack of manifestation in heterozygotes strongly argues for a mechanism other than loss of function in the previously reported Patients were evaluated ophthalmologically by one of the dominant mutations in these two genes. Thus, study of consanguineous authors (AOK). Written informed consent was obtained to re- populations has the additional advantage of not only identifying novel cruit patients (RAC #2070023). Sequencing of candidate genes recessive genes but also defining the mutational mechanism of dominant with or without homozygosity mapping was done as dictated by disorders. Genet Med 2011:13(11):978–981. the family history. Homozygosity mapping was performed by Axiom CEU Array (Affymetrix) genotyping according Key Words: sclerocornea, recessive mutations, homozygosity, inter- to the manufacturer’s protocol followed by analysis for runs of mediate fibers, BFSP2, PITX3, juvenile cataract homozygosity using autoSNPa as described before.3

he gene and disease paradigm has benefited greatly from the RESULTS Texpanding compendium of mutations and their effect on health. Key to the mechanistic understanding of this link be- The pedigrees of both study families are shown in Figure 1. tween mutations and the diseases they cause is the study of the Family 1 consists of healthy first cousin parents and one daughter molecular consequences of these mutations. Although an over- with bilateral severe congenital microphthalmia and a very severe all pattern can be appreciated in the mutational behavior of form of anterior segment dysgenesis (ASD) best described as many genes, e.g., mutations in enzyme-coding genes tend to sclerocornea (Fig. 2). An affected cousin was unavailable. Family behave recessively whereas multimeric structural -cod- 2 consists of healthy first cousin parents and three daughters with ing genes tend to behave dominantly, there are instances in juvenile-onset diffuse cortical cataract with scattered lens opacities which individual genes can harbor both classes of mutations. (age of symptoms approximately 12 years of age for each; Fig. 1). These genes are particularly interesting to study because the The father had had bilateral cataract surgery for posterior subcap- distinct pathogenic mechanisms for recessive and dominant sular cataract at 44 years of age. The 50-year-old mother and six other children were unaffected. For Family 1, we sequenced a panel of genes known to cause ASD (CYP1B1, PAX6, PITX2, and PITX3) and identified a homozygous From the 1Department of Genetics, King Faisal Specialist Hospital and Re- search Center; 2Department of Pediatric Ophthalmology, King Khaled Eye Spe- mutation in PITX3 c.640_656del (p.(Ala214ArgfsX42)) (Fig. 1). For cialist Hospital; 3Department of Anatomy and Cell Biology, College of Medicine, Family 2, we performed homozygosity scan and identified a single Alfaisal University; and 4Department of Pediatrics, King Khalid University Hospital run of a homozygosity shared between all affected patients. This is and College of Medicine, King Saud University, Riyadh, Saudi Arabia. a known dominant cataract that is linked to BFSP2 muta- Fowzan S Alkuraya, MD, Developmental Genetics Unit, Department of tions. Surprisingly, we identified a homozygous BFSP2 mutation Genetics, King Faisal Specialist Hospital and Research Center, MBC 03, PO c.598_599dup (p.(Ala201ArgfsX19)) (Fig. 1). Both PITX3 and Box 3354, Riyadh 11211, Saudi Arabia. E-mail: [email protected]. BFSP2 mutations were present in a heterozygous state in the The first two authors contributed equally to this work. unaffected parents. The BFSP2 mutation was also identified in Disclosure: The authors declare no conflict of interest. three of the six healthy children. Submitted for publication February 24, 2011. Accepted for publication May 24, 2011. DISCUSSION Published online ahead of print August 10, 2011. We report the identification of the first recessive mutations in DOI: 10.1097/GIM.0b013e31822623d5 two key regulators of lens development in : BFSP2 and

978 Genetics IN Medicine • Volume 13, Number 11, November 2011 Genetics IN Medicine • Volume 13, Number 11, November 2011 Novel Recessive BFSP2 and PITX3 Mutations

Fig. 1. Pedigrees of Family 1 and Family 2. Affected cousins (by history) in both families were unavailable.

PITX3. BFSP2 encodes CP49, an intermediate filament (IF) dominant negative fashion by interfering with the assembly of the protein that is lens-specific in its expression.4 Like other IF wild-type protein, particularly when one considers that all three in the lens, CP49 plays a critical role in the highly mutations affect the rod domain which is required for the beaded orchestrated organization of the of the differenti- filament assembly. The mutation we report here is almost certainly ated fiber cells.5 The hexagonal architecture of these fiber cells null because the mutant protein lacks most of the Coil1b domain provides a remarkably efficient packaging mechanism that re- and the entire rod domain and the downstream Coil-2 domain.12 duces intercellular spaces which is highly suited for the strict Therefore, we hypothesize that this mutant protein that com- optical properties of the lens.6 CP49 is an atypical IF protein, pletely lacks the ability to assemble or polymerize is unlikely however, in that it lack the C-tail seen in other IF.7 It does not to interfere with the wild-type protein. This is likely tolerated form homopolymers but rather heterodimerizes with BFSP1, by the lens fiber cells and may explain the lack of phenotypic another lens-specific IF protein.8 Mutations of both BFSP1 and consequences in the heterozygotes in this family (mother, BFSP2 are known to cause cataract in humans. grandparents, and three healthy children); however, we can- Three dominant mutations have been reported to date in BFSP2, not exclude that early onset senile cataract in the father, all involving single amino acids (two missense and one in-frame although different in morphology, may have been influenced deletion).9–11 The resulting phenotype was a cataract phenotype by his carrier status. Our hypothesis is also supported by the (juvenile and congenital) that is comparable with the one reported lack of phenotype in the mice that are heterozygous for here, but the pathogenic mechanism of those dominant mutations complete null alleles.13 Furthermore, the only human muta- remains unknown. The involvement of single amino acids makes it tion to date in the closest relative of BFSP2, BFSP1, is also tantalizingly plausible that the intact mutant protein acts in a a null mutation that causes cataract only recessively.14

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Fig. 2. A, Orbital CT shows microphthalmia in patient in Family 1. B, Slit-lamp examination of one of the patients in Family 2 showing cortical cataract with scattered lens opacities. C and D, Sequence chromatogram showing mutations in PITX3 and BFSP2, respectively.

Similarly, PITX3, which is known for its pivotal role in the being the last coding exon. The previously reported 17 bp normal formation of the lens vesicle and separation from the duplication results in a frameshift in codon 220 and produces lens ectoderm, is only known to cause ASD dominantly in an aberrant protein consisting of 94 additional residues, i.e., humans. Three mutations have been reported to date in this tran- the novel stop codon is introduced downstream to the orig- scription factor, but the pathogenic mechanism of the mutation is inal stop codon so nonsense-mediated decay is unlikely to be still unclear.15,16 For instance, one recent study of two of the three triggered. Similarly, our 17 bp deletion results in a frameshift dominant human mutations showed that the mutant PITX3 retains in codon 220, introduces 40 novel residues followed by a the capacity to localize to the nucleus and bind to the consensus stop. Because this premature stop codon is also in exon 3 promoter sequence.17 However, it seems that mutant PITX3 was which is the last coding exon, NMD would also be unlikely. unable to form homo or heterodimers. This, in addition to the Lack of any phenotype in the carrier parents of this patient documented reduced transcriptional activation by these mutant makes it highly unlikely that previously reported mutations proteins (40–70%), makes it tempting to speculate a dominant are simple loss of function. This may also explain why mice negative mechanism in which a PITX3 protein with reduced acti- heterozygous for null Pitx3 were phenotypically normal vation/transactivation capacity occupies the site of action of the compared with homozygotes.18 We posit, therefore, that at normal counterpart.17 The novel PITX3 mutation we report here least some instances of dominant human diseases which can involves deletion of the same 17 bp that are duplicated in a only be recapitulated in homozygous mouse models can be recurrent dominant mutation. It seems that both our deletion and explained by different mutational mechanisms between hu- the previously reported duplication are mediated by the presence of man and the mouse model rather than by difference in an 11 bp repeat.15 We note here that our recessive mutation results redundancy level between species. in a more severe phenotype (sclerocornea and microphthalmia) Consanguineous populations have been invaluable to the compared with the cataract/ASD phenotype described with domi- identification of recessively acting disease genes. In this study, nant mutations. Why deletion should act recessively while a and in another where we described the first recessively acting duplication should act dominantly is unclear but we note the CRYAB mutation,19 we demonstrate the potential of these pop- two frameshifting events create different novel amino acids ulations in improving our understanding the molecular mecha- for varying lengths at the C-terminus. PITX3 encodes a 302 nism of dominant disorders, an important step toward the design aa protein and comprises four exons with the third exon of molecular therapeutics.

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ACKNOWLEDGMENTS mutation in the beaded filament structural protein-2. Am J Hum Genet 2000;66:1426–1431. This study was funded by a grant from KACST (08- 10. Ma X, Li FF, Wang SZ, Gao C, Zhang M, Zhu SQ. A new mutation in MED497-20) and a Collaborative Research Grant from DH- BFSP2 (G1091A) causes autosomal dominant congenital lamellar cataracts. FMR to FSA. We sincerely thank the patients and their families Mol Vis 2008;14:1906–1911. for their enthusiastic participation. 11. Jakobs PM, Hess JF, FitzGerald PG, Kramer P, Weleber RG, Litt M. Autosomal-dominant congenital cataract associated with a deletion mutation in the human beaded filament protein gene BFSP2. Am J Hum Genet 2000;66:1432–1436. 12. Merdes A, Gounari F, Georgatos SD. The 47-kD lens-specific protein REFERENCES phakinin is a tailless intermediate filament protein and an assembly partner of filensin. J Cell Biol 1993;123:1507–1516. 1. Antonarakis SE, Cooper DN. Human gene mutations: mechanisms and 13. Sandilands A, Prescott AR, Wegener A, et al. Knockout of the intermediate consequences, 4th ed. Berlin: Springer, 2010. filament protein CP49 destabilises the lens fibre cell cytoskeleton and 2. Aldahmesh MA, Safieh LA, Alkuraya H, et al. Molecular characterization of decreases lens optical quality, but does not induce cataract. Exp Eye Res retinitis pigmentosa in Saudi Arabia. Mol Vis 2009;15:2464–2469. 2003;76:385–391. 3. Carr IM, Flintoff KJ, Taylor GR, Markham AF, Bonthron DT. Interactive 14. Ramachandran RD, Perumalsamy V, Hejtmancik JF. Autosomal recessive visual analysis of SNP data for rapid autozygosity mapping in consanguin- juvenile onset cataract associated with mutation in BFSP1. Hum Genet eous families. Hum Mutat 2006;27:1041–1046. 2007;121:475–482. 4. Georgatos SD, Gounari F, Remington S. The beaded intermediate filaments 15. Semina EV, Ferrell RE, Mintz-Hittner HA, et al. A novel gene and their potential functions in eye lens. Bioessays 1994;16:413–418. PITX3 is mutated in families with autosomal-dominant cataracts and 5. Perng MD, Zhang Q, Quinlan RA. Insights into the beaded filament of the ASMD. Nat Genet 1998;19:167–170. eye lens. Exp Cell Res 2007;313:2180–2188. 16. Berry V, Yang Z, Addison PK, et al. Recurrent 17 bp duplication in PITX3 6. Song S, Landsbury A, Dahm R, Liu Y, Zhang Q, Quinlan RA. Functions of is primarily associated with posterior polar cataract (CPP4). J Med Genet the intermediate filament cytoskeleton in the eye lens. J Clin Invest 2009; 2004;41:e109. 119:1837–1848. 17. Sakazume S, Sorokina E, Iwamoto Y, Semina EV. Functional analysis of 7. Perng MD, Quinlan RA. Seeing is believing! The optical properties of the human mutations in homeodomain PITX3. BMC Mol eye lens are dependent upon a functional intermediate filament cytoskeleton. Biol 2007;8:84. Exp Cell Res 2005;305:1–9. 18. Ho HY, Chang KH, Nichols J, Li M. Homeodomain protein Pitx3 8. Carter JM, Hutcheson AM, Quinlan RA. In vitro studies on the assembly maintains the mitotic activity of lens epithelial cells. Mech Dev. 2009; properties of the lens proteins CP49, CP115: coassembly with alpha-crys- 126:18–29. tallin but not with vimentin. Exp Eye Res 1995;60:181–192. 19. Safieh LA, Khan AO, Alkuraya FS. Identification of a novel CRYAB 9. Conley YP, Erturk D, Keverline A, et al. A juvenile-onset, progressive mutation associated with autosomal recessive juvenile cataract in a Saudi cataract locus on 3q21–q22 is associated with a missense family. Mol Vis 2009;15:980–984.

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