|Molecular Vision 2007;
Received 5 May 2007 | Accepted 12 September 2007 | Published 13 September 2007
Novel mutations of the FRMD7 gene in X-linked congenital motor nystagmus
Shanhu Xu,1 Qian
Li,3 Fei Song,1
Jinfu Yin,2 Kun
Xia,3 Jiahui Xia3
1Department of Neurology, and 2Department of Ophthalmology, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China; 3National Laboratory of Medical Genetics of China, Central South University, Changsha, Hunan, China
Correspondence to: Baorong Zhang, M.D., Department of Neurology, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, People's Republic of China; Phone: +86-571-87784752; FAX: +86-571-87784750; email: firstname.lastname@example.org
Purpose: Congenital motor nystagmus (CMN) is a relatively common oculomotor disorder characterized by bilateral uncontrollable ocular oscillations. Recently, the FRMD7 gene mutation has been identified as the genetic cause of CMN. The purpose of this study was to identify mutations of the FRMD7 gene in Chinese patients with CMN.
Methods: Clinical data and genomic DNA of three Chinese CMN families were collected after informed consent. Genescan by two-point linkage analysis combined with haplotype analysis was performed and mutation screening of the FRMD7 gene was conducted by direct sequencing.
Results: Maximum two-point LOD scores of 2.00, 1.76, and 1.16 at θ=0.00 were obtained with markers in proximity to the FRMD7 gene on chromosome Xp26 in the three CMN families. Mutation screening in the FRMD7 gene identified two novel missense mutations (c.781C>G and c.886G>C) and one reported nonsense mutation (c.1003C>T). These nucleotide alterations were not seen in unaffected members of the families or in 100 unrelated control subjects.
Conclusions: This study widens the mutation spectrum of the FRMD7 gene.
Congenital motor nystagmus (CMN), also termed idiopathic congenital nystagmus, is a common hereditary disorder characterized by bilateral ocular oscillations that occur in the first 6 months of life [1-3]. The prevalence of CMN in the human population is estimated to be between 1:1,000 and 1:1,500 [4,5]. CMN is distinct from other genetic ocular disorders in which nystagmus accompanies a clinically apparent defect in the visual sensory system . CMN could cause visual impairment and visual acuity can be diminished, usually slightly to moderately. The mechanism of CMN remains unclear and it is presumed to be secondary to an abnormal development of those ocular motor areas of the brain that control fixation [2,7,8]. There is no curative treatment currently for the disease [9,10]. CMN can be inherited in various patterns and X-linked inheritance with incomplete penetrance and variable expressivity is probably the most common form. CMN with X-linked inheritance has been mapped to two regions: Xp11.4-p11.3  and Xq26-q27 [2,11-13]. The mutation in the FRMD7 gene (Genbank NM_194277) is the major cause of familial X-linked CMN mapped to Xq26-q27 [14,15]. We recently collected three CMN families. To confirm whether the FRMD7 gene was mutated in these families, we undertook linkage analysis and mutation analysis of the FRMD7 gene in the three CMN families.
Three Chinese families (families 1, 2, and 3) with CMN were from Zhejiang province, in which there were 50 family members including 11 affected males, 12 affected females, and 27 unaffected individuals (Figure 1). Family 1 was previously reported by us . All patients were clinically diagnosed at the Second Affiliated Hospital of Zhejiang University after detailed ocular-neurological examination [1,8]. The fundus photographs were recorded by a TRC.50EX Retinal camera (Topcon Corp, Tokyo, Japan) and the electroretinograms were recorded on an LKC, UTAS-3000 (LKC Technologies Inc., Gaithersburg, MD). All members were recruited with informed consent and with appropriate local and regional ethics review committee approvals. Blood samples were obtained from these family members and DNA was prepared using standard methods. Control DNAs (n=100) from ethnically matched apparently healthy adults were anonymous.
Genotyping and linkage analysis
Microsatellite markers covering the short and long arm of the X chromosome were tested using fluorescent labeled primers, and all these markers were previously used for fine mapping of X-linked CMN [2,11,12]. Alleles were analyzed by GENESCAN Analysis version 3.0 and GENOTYPER version 2.1 software. Two-point LOD scores were calculated by the MLINK program of the LINKAGE package (version 5.1). Linkage analysis was performed for these families as previously described . The disease was specified to be an X-linked dominant trait with penetrances of 0.9 and 1.00 in females and males, respectively. The allele frequency was assumed to be equal, as well as the recombination frequencies in males and females. We assumed gene frequencies of 0.0001 and no sex difference in recombination.
We designed 17 genomic amplicons to cover the FRMD7 gene coding region and some flanking intronic sequences in each case. PCR primers were designed by the primer3 online software and the sequences presented in Table 1. For all amplicons, 30 ng genomic DNA was amplified in a volume of 10 μl containing 10X Qiagen HotStar Taq buffer, 1.5 mM MgSO4, 0.2 mM dNTP, 2 μl Q solution, 0.5 μl forward primer, 0.5 μl reverse primer, 4.3 μl ddH2O and 0.05 U of HotStar Taq. Thermal cycling was performed using a Perkin Elmer Corporator thermal cycler (Applied Biosystems, Foster City, CA). PCR conditions were: 1 cycle of 95 °C for 15 min; 12 cycles of 94 °C for 30 s, 63 °C for 45 s, and 72 °C for 60s; 25 cycles of 94 °C for 30 s, 56 °C for 45 s, and 72 °C for 60 s; and 1 cycle of 72 °C for 10 min.PCR products were digested with Exonuclease I and Shrimp Alkaline Phosphatase to remove the free primers, and both strands were sequenced on an ABI-PRISM 3130 automatic sequencer (Applied Biosystems).
Sequencing results were assembled and analyzed using the SeqMan II program of the Laser gene package (DNA STAR Inc., Madison, WI). For all samples containing an abnormal FRMD7 amplicon, new PCR products were reamplified from genomic DNA using the same protocols. Mutations identified were confirmed on new independent samples. The sequence variants are numbered according to the GenBank reference cDNA sequence NM_194277 with the "A" of the ATG translation initiation codon being nucleotide 1. To determine the evolutionary conservation of identified substitutions, the ExPASy proteomics server was used to look for homologues of the FRMD7 protein.
In families 1, 2, and 3, an X-linked inheritance pattern was identified. The disease was clearly transmitted from female carriers to affected sons. No male to male transmission was found. The penetrance within these families varied considerably in the female carriers but was consistently complete in the male offsprings. Penetrance was from 54% to about 100% among obligate female carriers (daughters of affected men; family 1, three of three; family 2, two of four; family 3, one of two). The results of the ophthalmologic and neurological system examination were normal. Normal color vision was recorded in all affected individuals. Fundus photograph and electroretinogram examinations were normal in the three individuals tested (family 1, III: 14; family 2, IV: 4; family 3, III: 2; Table 2).
We found no evidence of linkage within the chromosome Xp in Families 1, 2, or 3. However, linkage was initially established without recombination with seven Genethon markers between the regions Xq26-27 closely linked to the FRMD7 gene. The maximal LOD scores of 2.00, 1.76, and 1.16 were obtained in families 1, 2, and 3 on chromosome Xq26, respectively (Table 3). A recombination event in an affected male in Family 2 refined the location of the CMN gene between markers DXS8009 and DXS8094 (Figure 1).
We identified two novel missense mutations and one reported nonsense mutation in the FRMD7 gene by direct sequencing of the coding and partial intron regions. In family 1, all affected members had the missense mutation c.781C>G in exon 9 (Figure 2A); in family 2, all the patients and obligate carriers had the missense mutation c.886G>C in exon 9 (Figure 2B); in family 3, a previously reported nonsense mutation c.1003C>T in exon 11  was detected (Figure 2C). Obligate female carriers were heterozygous in these mutations and the affected males were homozygous, consistent with X-linked inheritance. All mutations identified above cosegregated with the disease in the families and were absent in the 100 control subjects. The results of the ExPASy proteomics server indicated that the three mutations were within the IMP dehydrogenase/GMP reductase domain (Figure 3).
In this study, three families (Families 1, 2, and 3) had high LOD scores suggesting linkage to chromosome Xq, which contains the FRMD7 gene identified as the cause of CMN. Three mutations, including two novel missense mutations and one reported nonsense mutation, were identified. The three mutations resulted in amino acid substitutions at p.R261G, p.G296R and p.R335X, respectively. These mutations are highly conserved residues that are invariant in Mus musculus, Macaca mulatta, Gallus gallus, Rattus norvegicus, and Canis familiaris, suggesting that mutations at these locations are functionally significant to the protein (Figure 3).
The mutations identified in this study widen the mutation spectrum of the FRMD7 gene and this study supports that mutations in the FRMD7 gene are the major cause of X-linked congenital motor nystagmus as described by Tarpey et al.  The mutations identified scattered in almost all the exons and the splice sites as showed in Figure 4. However, the major mutations are clustered at the NH2-terminus, where homology to B41 and FERM-C domain is present . The COOH-terminus of FRMD7 (amino acid residues 280-714) has no significant homology to other database entries and contains unknown functions. This is thought to be a newly identified member of the FERM family . FERM domains define the band 4.1 superfamily and FERM family members provide a link between the membrane and cytoskeleton and are involved in signal transduction pathways. The B41 protein play structural and regulatory roles in the assembly and stabilization of specialized plasmamembrane domains [17-19]. FERM-C domain is the third structural domain within the FERM domain . In order to predict the possible effects of the various mutations on the protein characteristics, we analyzed the motif of the protein using the ExPASy proteomics server. The results of the ExPASy proteomics assay indicate these mutations occur within the IMP dehydrogenase/GMP reductase domain, which is involved in biosynthesis of guanosine nucleotide . However there is no definitive relationship between the function of IMP dehydrogenase/GMP reductase domain and the mechanism of X-linked CMN. Additional experiments are required to investigate whether a general structure of the protein or specific alterations within possible interacting domains is causing the reduction in function of the various mutated forms of the protein.
The true impact of genetic defects in the FRMD7 gene and the variable clinical expression of CMN within and between families with the same FRMD7 gene mutations require further investigation to elucidate the function of this protein. The two novel mutations that we detected in the Chinese population widen the FRMD7 gene mutation spectrum. It is hoped that the identification of FRMD7 mutations underlying CMN will enable a more rapid molecular diagnosis and deeper understanding of the pathological mechanisms of CMN.
The authors would like to thank the families for their enthusiasm and participation in this study. This study was supported by grants from the National Natural Science Foundation of China (30670742 to BZ). We also thank Professor Ming Qi for critical reading this manuscript and Mr. Hao Zhang for assistance in DNA preparation.
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