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. Genes 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 gene 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 microphthalmia. 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 Human 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 protein-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 locus 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 humans: 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 proteins 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 cytoskeleton 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 Genetics IN Medicine • Volume 13, Number 11, November 2011 979 Aldahmesh et al. Genetics IN Medicine • Volume 13, Number 11, November 2011 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