January 2005
Volume 46, Issue 1
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Biochemistry and Molecular Biology  |   January 2005
Truncating Mutation in the NHS Gene: Phenotypic Heterogeneity of Nance-Horan Syndrome in an Asian Indian Family
Author Affiliations
  • Vedam Lakshmi Ramprasad
    From the Department of Genetics and Molecular Biology, Vision Research Foundation, Sankara Nethralaya, Chennai, India; the
  • Alka Thool
    Shri Ganapati Nethralaya, Jalna, Maharashtra, India; the
  • Sakthivel Murugan
    From the Department of Genetics and Molecular Biology, Vision Research Foundation, Sankara Nethralaya, Chennai, India; the
  • Derek Nancarrow
    Queensland Institute of Medical Research, Brisbane, Australia; the
  • Prateep Vyas
    Shri Ganapati Nethralaya, Jalna, Maharashtra, India; the
  • Srinivas Kamalakar Rao
    Department of Cataract and IOL Implantation, Medical Research Foundation, Sankara Nethralaya, Chennai, India.
  • Authiappan Vidhya
    From the Department of Genetics and Molecular Biology, Vision Research Foundation, Sankara Nethralaya, Chennai, India; the
  • Krishnamoorthy Ravishankar
    Department of Cataract and IOL Implantation, Medical Research Foundation, Sankara Nethralaya, Chennai, India.
  • Govindasamy Kumaramanickavel
    From the Department of Genetics and Molecular Biology, Vision Research Foundation, Sankara Nethralaya, Chennai, India; the
Investigative Ophthalmology & Visual Science January 2005, Vol.46, 17-23. doi:10.1167/iovs.04-0477
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      Vedam Lakshmi Ramprasad, Alka Thool, Sakthivel Murugan, Derek Nancarrow, Prateep Vyas, Srinivas Kamalakar Rao, Authiappan Vidhya, Krishnamoorthy Ravishankar, Govindasamy Kumaramanickavel; Truncating Mutation in the NHS Gene: Phenotypic Heterogeneity of Nance-Horan Syndrome in an Asian Indian Family. Invest. Ophthalmol. Vis. Sci. 2005;46(1):17-23. doi: 10.1167/iovs.04-0477.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. A four-generation family containing eight affected males who inherited X-linked developmental lens opacity and microcornea was studied. Some members in the family had mild to moderate nonocular clinical features suggestive of Nance-Horan syndrome. The purpose of the study was to map genetically the gene in the large 57-live-member Asian-Indian pedigree.

methods. PCR-based genotyping was performed on the X-chromosome, by using fluorescent microsatellite markers (10-cM intervals). Parametric linkage analysis was performed by using two disease models, assuming either recessive or dominant X-linked transmission by the MLINK/ILINK and FASTLINK (version 4.1P) programs (http:www.hgmp.mrc.ac.uk/; provided in the public domain by the Human Genome Mapping Project Resources Centre, Cambridge, UK). The NHS gene at the linked region was screened for mutation.

results. By fine mapping, the disease gene was localized to Xp22.13. Multipoint analysis placed the peak LOD of 4.46 at DSX987. The NHS gene mapped to this region. Mutational screening in all the affected males and carrier females (heterozygous form) revealed a truncating mutation 115C→T in exon 1, resulting in conversion of glutamine to stop codon (Q39X), but was not observed in unaffected individuals and control subjects.

conclusions. A family with X-linked Nance-Horan syndrome had severe ocular, but mild to moderate nonocular, features. The clinical phenotype of the truncating mutation (Q39X) in the NHS gene suggests allelic heterogeneity at the NHS locus or the presence of modifier genes. X-linked families with cataract should be carefully examined for both ocular and nonocular features, to exclude Nance-Horan syndrome. RT-PCR analysis did not suggest nonsense-mediated mRNA decay as the possible mechanism for clinical heterogeneity.

Congenital cataract can be isolated or a part of a syndrome. 1 The familial occurrence of isolated cataract, modes of inheritance, and genes involved have been well described. Reports typically indicate an autosomal dominant mode of transmission, and the implicated genes include crystallins, connexins, and others. 2 There are many reports of autosomal recessive forms of congenital cataract, 3 whereas only a few on X-linked inheritance have been published. A recent report 4 describes the occurrence of X-linked recessive, nonsyndromic, developmental cataract in a family; however, several affected males in this family also had cardiac defects. Because none of the affected males in the pedigree had offspring, the investigators concluded that they could not confirm the recessive nature of the X-linked transmission. 4  
There has been particular interest regarding X-linked cataract, since it is one of the features of X-linked syndromes such as Nance-Horan syndrome. 5 To date, there have been few gene-mapping studies on X-linked Nance-Horan syndrome. 6 7 The syndrome, also referred to as the cataract–dental syndrome, is characterized in males by ocular manifestations of congenital cataract, microcornea, early-onset nystagmus, and nonocular manifestations of anteverted pinnae, short fourth metacarpal bones (brachymetacarpalia), and multiple dental anomalies. The dental abnormalities include maxillary and mandibular diastema of both central and lateral incisors, notched or serrated incisal edges, and screwdriver-shaped teeth due to narrow gingival and incisal margins. Characteristic dysmorphic features include a long, narrow face and prominent nose and nasal bridge. 6 Mental impairment is also a clinical component of Nance-Horan syndrome. 8 Lewis et al. 6 studied five affected families, and they reported that 100% of the affected males had severe congenital cataract, microcornea, nystagmus, and dental abnormalities; 95% had short lateral metacarpals; and 76% had anteverted pinnae. Of the carrier females, 100% had posterior Y-sutural cataracts, small corneas, and dental anomalies; 95% had short lateral metacarpals; and 19% had anteverted pinnae. 
Investigators used linkage studies to localize the gene to the Xp22 region. 6 A study of 13 independent multiplex families affected with classic Nance-Horan syndrome localized the gene responsible to the Xp22.13 region. 9 Studies on the extended Australian family, in which Horan and Bilson first identified Nance-Horan syndrome, confirmed localization of the disease gene to a 1.3-Mb interval at Xp22.13. 10 Gene-mapping studies of Nance-Horan syndrome, X-linked cataract with microcornea, and X-linked nuclear cataract (with a few males having cardiac defects) and the present study map the disease loci to Xp22.31-p22.13. The NHS gene was localized to this region, and mutations were identified within the coding region of the gene. 10  
In this report, we describe a four-generation family with multiple affected individuals who were reported to have developmental lens opacities transmitted in a manner consistent with an X-linked recessive mode of inheritance (Fig. 1) . All eight affected males had severe congenital cataract and microcornea, which was noted on the first visit. While performing linkage analyses, we realized the proximity of the disease locus to the recently identified NHS gene, which has been identified recently. Afterward, we recalled and re-examined the family and noted that the clinical severity of other features of Nance-Horan syndrome was either mild or varied highly in some of the members; hence, the NHS gene was screened for identifying mutation. 
Methods
Clinical Examination
The institutional review board (Sankara Nethralaya) approved the project, and the work adhered to the tenets of the Declaration of Helsinki. A 45-year-old man (Fig. 1 , individual 14), who was aphakic in both eyes, presented to us with complaints of decreased vision in the right eye. He had best spectacle-corrected visual acuity (BSCVA) of counting fingers and nystagmus in both eyes. He also had microcornea, and there was dense posterior capsule opacification in both eyes. Posterior segment evaluation showed the presence of a retinal detachment in the right eye. Successful repair of the retinal detachment resulted in a BSCVA of 20/400 in the right eye. During examination, the proband (Fig. 1 , individual 14) reported the occurrence of developmental lens opacities in males in the family and thus three generations, 57 live members (Fig. 1)of the family were examined. An ophthalmologist performed the clinical examination, a history of associated systemic features was noted in a questionnaire, and a physician examined the subjects to rule out systemic abnormalities, such as dental anomalies, unusual facial features, and mental retardation. In cataractous males who consented, echo and electrocardiogram were performed to exclude cardiovascular defects and radiography to rule out brachymetacarpalia. Congenital cataract, with or without microcornea with visual morbidity, was the minimum diagnostic criterion for affected males (Table 1) . A female was classified as a carrier based on her position in the pedigree with an affected father and with mild to less severe lens opacities (sutural/blue dot). 6 Blood samples were collected from 57 individuals for DNA extraction and genetic analysis. 
Genotyping
PCR-based microsatellite marker genotyping was performed by using dinucleotide fluorescent markers on the X chromosome at 10-cM intervals (panel 28 of the Prism linkage mapping set, ver. 2.5; Applied Biosystems [ABI], Inc., Foster City, CA). Genotyping was performed on a genetic analyzer (PRISM 310; ABI) with accompanying software (GeneScan; ABI). Four additional fluorescence-labeled dinucleotide markers on Xp22.31-p22.13 (tel-DXS7104-DXS1196-DXS7163-DXS999-cen) were used for fine mapping. 
Linkage Analysis
Two-point and multipoint LOD scores were calculated using the MLINK/ILINK programs and FASTLINK package (ver. 4.1P; http:www.hgmp.mrc.ac.uk/; provided in the public domain by the Human Genome Mapping Project Resources Centre, Cambridge, UK). 11 12 13 Analysis assumed an X-linked recessive transmission with complete penetrance, a co-dominant mode of inheritance with 99% penetrance, and a gene frequency of 0.0001. The disorder also appears to be inherited in a co-dominant fashion, with heterozygous females manifesting the disease but having less severe clinical features than males—hence, the need for analyses by both types of linkage models. Equal marker allele frequencies were used, with the segregating allele frequency for each marker varying from 0.125 to 0.25, depending on the number of alleles present within the pedigree. Multipoint analysis was performed, using the Marshfield sex-averaged, intermarker map distances of 1.91, 0.54, 0.54, 0.0, and 4.33 cM between the six fine-mapping markers DXS7104, DXS987, DXS1195, DXS999, DXS7163, and DXS1226, respectively. The correct orientation of DXS999 and DXS7163 was obtained using the Ensemble Genome Browser (ensembl: http://www.ensembl.org). 14  
Mutational Screening
We screened the family for mutations in the NHS gene, by PCR amplification and direct DNA sequencing (Prism 310 Genetic analyzer; ABI) for the affected males and carrier females and computer analysis (Sequence software, ver. 1.11; ABI). NHS gene screening was also performed in a few control subjects from within the family (Fig. 1 ; individuals 6, 10, 15, 17, 19, 23, 26, 29, 34, and 37). Nineteen sets of primers, which include intronic and overlapping exonic regions, were used for amplification of all nine exons (all primer sequences were provided by Jamie E. Craig, Menzies Center for Population Health Research, University of Tasmania, Australia). The amplified products were electrophoresed in 2% agarose gel for verification and followed by sequencing. 
RT-PCR Analysis
To investigate the possibility that this exon-1 mutation might lead to nonsense-mediated decay, RNA was isolated from the lymphocytes separated from 10-mL heparinized blood samples of three affected males, one unaffected male within the family, and one unrelated unaffected male, by using an extraction reagent (TRI; Sigma-Aldrich, St. Louis, MO), according to the recommendations of the manufacturer, and dissolved in diethyl pyrocarbonate (DEPC)–treated water. DNase-treated RNA, cloned Moloney murine leukemia virus (MMLV) reverse transcriptase (USB, Cleveland, OH), and random hexamers (Amersham Biosciences, Piscataway, NJ) were used to generate a cDNA pool by reverse transcription–polymerase chain reaction (RT-PCR). A no-RT control was also run. PCR primers for the ABL housekeeping gene were used as the internal control, and the primers were designed to check genomic DNA contamination (forward 5′-GGCCAGTAGCATCTGACTTTG-3′ and reverse 5′-ATGGTACCAGGAGTGTTTCTCC-3′). These primers span an intron and amplify a product of 859 bp from contaminating DNA and 296 bp from the expected cDNA. For the amplification of the ABL gene fragment PCR was performed at 94°C for 10 minutes followed by 35 cycles of 94°C for 1 minute 15 seconds, 60°C for 1 minute 30 seconds, and 72°C for 2 minutes followed by a final extension of 72°C for 10 minutes. For the amplification of the NHS gene, two pairs of exonic primers 10 were used: one for major isoform A (62 bases downstream of the mutation site in exon 1, forward 5′-GCCGTCCCTGCACCTTCA-3′ and reverse 5′-GTTGCTGACCGCGCATAG-3′) and the other for both isoforms A and B (region in exon 6, forward 5′-TCCCCGGGAAGGTAATAGAG-3′ and reverse 5′-TGAGGGGCTGTGTTTAGTGA-3′). PCR amplification for the minor isoform B was performed using 50 ng of cDNA at 94°C for 10 minutes followed by 35 cycles of 94°C for 1 minute, 56°C for 1 minute, 72°C for 2 minutes, and a final extension of 72°C for 10 minutes. For the major isoform A, PCR was performed with a touchdown protocol with 2 M betaine in the reaction mixture, because the region is GC rich. Touchdown annealing temperatures started at 64°C and ended at 59°C (ΔT of −0.5°C per cycle) for 11 cycles, followed by 25 cycles at 59°C. No-template PCR controls were included in all PCR runs and were always negative. All the amplified products were run on 2% agarose gel with ethidium bromide. 
Results
Clinical Examination
The parents of the proband were deceased, but the history revealed that the proband’s mother (Fig. 1 , individual 2) had been affected with congenital cataract and that his father (Fig. 1 , individual 1) had been normal. Details of the clinical examination of the other members are given in Table 1 . The clinical findings indicated that the disease, especially lens opacity, are significant in the males, resulting in early cataract surgery. Postoperative poor visual acuity and nystagmus may reflect the severity of the lens change, delay in surgery, or the success of amblyopia therapy after cataract surgery. Retinal detachments were present in four of the eyes in these patients, but the absence of peripheral retinal changes in the fellow eyes and the vitreous disturbance associated with cataract surgery indicate that these are probably secondary to surgery and do not represent a primary retinal degeneration. All affected males except individual 52 (horizontal and vertical corneal diameters, 10.6 mm each) had distinct microcornea, with corneal diameters ranging between 8 and 10 mm. A few female carriers had reduced corneal diameters (ranging from 10 to 11 mm; Table 1 ). The typical dental anomalies of Nance-Horan syndrome were not observed; however, mild to moderate expression of the phenotype was noted in affected males 14 (not shown) and 52 (Figs. 2B 5D)and carrier female 32 (Fig 2C) . Individuals 3, 7 (not shown), and 16 (Fig. 2E)did not have any dental anomalies; however, individual 3 who was 64 years of age, had lost some of the incisor and canine teeth due to old age. Individuals 7 and 14 had fibrosis of the oral mucosa, which could have been due to constant tobacco chewing. Anteverted pinnae were observed in individual 52 (Fig. 2B) . No family members exhibited a long, thin face or the thin nasal bridge associated with Nance-Horan syndrome although many had a prominent nose (including individual 23, who married into this family). Individual 7 was dysmorphic, with mandibular prognathism and some degree of midfacial retrusion. Affected males 3 and 7 and carrier female 45 had moderate to mild facial asymmetry. Brachymetacarpalia, cardiovascular abnormalities, and mental retardation were not observed in the family. 
Summarizing the findings in this family, there was no transmission of the disease from father to son but there was transmission of the disease from affected and unaffected mothers to sons, suggesting X-linked recessive inheritance (Fig. 1) . Affected fathers also had daughters with a milder version of the disease, such as blue dot or cortical changes in the lens or both, in all the carrier females (Table 1) . All affected second-generation males had had cataract extraction, except for individual 16 who had had a combination of sutural, coralliform, blue dot, and posterior subcapsular opacities. Lens changes in the fourth-generation subjects include posterior polar changes (Fig. 1 ; individual 52), blue-dot opacity (Fig. 1 , individual 57), and cortical cataract (Fig. 1 , individual 61), which resulted in a BSCVA ranging from 20/120 to 20/40. Subjects 52 and 56 (Fig. 1)had surgery at a very early age (4 and 3 months, respectively) compared with individual 60. Carrier females also had lens opacities, but none of them necessitated cataract surgery (Table 1)
Linkage Analysis
Linkage to 16 markers spanning the entire X chromosome was excluded. Initial positive LOD scores were obtained for markers DXS987 and DXS1226 (Table 2) . After fine mapping with additional markers (DXS7104, DXS1195, DXS999, DXS7163), linkage and haplotype analyses revealed that the disease locus is centromeric to DXS987 and telomeric to DXS7163. Linkage analysis using DXS1195 which maps between the above two markers is an intragenic marker of the NHS gene 10 that provided an LOD score of 2.11 at θ = 0 (Table 2)for the recessive and 2.69 for the dominant models. The two-point LOD scores for DXS999 and DXS7163 were both >3 (3.57 and 3.27, respectively), assuming dominant inheritance, and provided significant evidence of linkage; however, no critical recombinants were observed among DXS987, DXS1195, DXS999, and DXS7163 (Fig. 1)for either transmission model. The multipoint analysis, using the Marshfield genetic map and the dominant disease model, revealed a significant peak LOD of 4.46 at DXS987, although the entire 1-cM region between DXS987 and DXS999 maintained an LOD greater than 4.4 in the dominant model and 2.68 when the recessive model was used. These data are consistent with the pedigree segregation analysis shown in Figure 1 , which shows that all affected male individuals and female carriers, respectively, shared the disease haplotype 3-4-4-2 for DXS987, DXS1195, DXS7163, and DXS999. According to Ensemble, 14 this region encompasses a ∼4-Mb region of the X chromosome, which spans the Xp22.22 and Xp22.13 junction. 
Mutational Screening
All nine exons of the NHS gene were screened for mutations. A novel nonsense mutation, 115C→T in exon 1 was detected, resulting in conversion of glutamine to a stop codon (Q39X; Fig. 3 ; GenBank accession number AY633488; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD). This mutation was detected in all the affected males and in carrier females (heterozygous form) and was not detected in individuals 6, 10, 15, 17, 19, 23, 26, 29, 34, and 37 (Fig. 1)confirming the sequence change as a disease-causing, cosegregating mutation. 
RT-PCR Analysis
Total RNA was extracted from five male individuals (three affected and one unaffected pedigree members, as well as one unrelated, unaffected male) and used to generate cDNA. Amplicfication of a transintronic fragment of the housekeeping gene ABL showed only a 296-bp band for each sample, demonstrating that none of the five cDNAs were contaminating with genomic DNA (Fig. 4) . RT-PCR of the NHS gene revealed the expression of both major and minor isoforms in each of the five samples tested (Fig. 4) . The amplification of cDNA for isoform A showed no visible difference in expression levels between patients and control subjects (Fig. 4) . Thus, we could find no evidence of nonsense-mediated decay as a result of the exon-1 mutation segregating in this family. 
Discussion
By linkage analysis, we localized the disease region near the NHS locus with an LOD score of Z max = 3.57 for DXS999, assuming dominant transmission. Multipoint analysis places the LOD for the entire 1-cM region between DXS987 and DXS7163 higher than 4.4. A peak single-point LOD score of 2.41 for DXS999 and DXS7163 and a multipoint LOD of 2.68 spanning the region between DXS987 and DXS999 were obtained assuming recessive inheritance with complete penetrance. Recently, a positional cloning approach was used to identify a novel gene, called NHS, 10 in this critical region, and mutations were reported in five of six typical Nance-Horan–affected families (Fig. 5) . NHS is a large gene that encodes a 1630-amino-acid major isoform A protein and a 1335-amino-acid minor isoform B protein, which begins translation in exon 4. 10 NHS has an important regulatory role in the development of ocular, craniofacial, and neural tissue. It has been demonstrated that NHS protein is expressed in midbrain, lens, tooth, and retina. 10 The fact that the NHS gene is highly conserved across vertebrate species supports the vital role of the gene in development. 10  
In the present study, mutational screening of the NHS gene revealed a novel truncating mutation, 115C→T, in exon 1, which resulted in the conversion of glutamine to a stop codon (Q39X) in all the affected males and carrier females (heterozygous form). This mutation was not observed in unaffected individuals and related control subjects. RT-PCR demonstrated no evidence of nonsense-mediated decay in response to this mutation (Fig. 4) ; thus, it is predicted to result in the loss of a functional protein for major isoform A but not to alter minor isoform B, which begins in exon 4. However Burdon et al. 10 identified a truncating mutation in exon 1 (Arg134fs) in a family with features typical of Nance-Horan syndrome. Further analysis involving multiple NHS mutations is needed to explain why a truncated mutation at position 39 of a 1630-amino-acid sequence results in a mild to moderate nonocular phenotypic expression. This fact could support the role of modifier genes in nonocular tissues. 
In our first contact with the affected family, individuals presented with cataract, microcornea, and retinal detachment; however, our initial investigations did not detect the presence of subtle nonocular features. Nonetheless on finding linkage to Xp22.13 we suspected that this could be Nance-Horan syndrome and therefore recalled the family for complete re-examination. Among the pedigree members re-examined thoroughly for both ocular and systemic features of Nance-Horan syndrome, none had brachymetacarpalia or mental retardation. However, a few of the affected males (Fig. 1 , individuals 7 and 16) and carrier females (Fig. 1 , individuals 13, 28, and 45) had neither dental anomalies nor anteverted pinnae. In contrast, only individual 52 had anteverted pinnae (Fig. 2B) . A few pedigree members were not available for the re-examination of nonocular features; however all the observations collectively suggest the presence of Nance-Horan syndrome, with most of the affected pedigree members having profound ocular features but few having mild to moderate nonocular complications. This suggests that allelic heterogeneity within the NHS gene could manifest as severe cataract and microcornea but mild nonocular Nance-Horan syndrome features, perhaps indicating a role for modifier genes in nonocular tissues. We also believe that the occurrence of blue dot lens changes in some individuals (Fig. 1 , individuals 6 and 29) in this pedigree represents an independent change and does not reflect the effect of the NHS gene, which is responsible for the Nance-Horan syndrome in the other members. 
The data presented herein provide a strong case for a thorough physical examination of all the family members before a diagnosis of nonsyndromic familial cataract is made. We emphasize that all X-linked families with cataract should be carefully examined for further ocular and nonocular features, to exclude Nance-Horan syndrome, because nonocular features may be present in a subtle manner. In addition, we suggest that the NHS gene should be screened for mutations or the region between DXS987 and DXS7163 should be excluded before proceeding with further gene mapping. 
This work adequately provides evidence of a genotype–phenotype correlation, such that the Q39X mutation appears to manifest as a distinct expression of Nance-Horan features. It is interesting to note that, in case of diseases such as X-linked Wiskott-Aldrich syndrome (WAS) 15 missense mutations in exons 1 to 3 (the PH domain) of the WAS gene inhibit less-important functions of the protein and result in a mild phenotype, whereas mutations affecting exon 4 or beyond and the 3′ portion of the WAS gene interfere with the crucial function of the protein and thus cause classic WAS. 15 Because the NHS protein has not been characterized, further research into the structural and functional characteristics of the protein and its expression in ocular and nonocular tissues is needed to support this suggestion. Burdon et al. 10 also suggest the genetic heterogeneity of the syndrome, as they had a family without any mutation but with the classic features of Nance-Horan syndrome. Further research into Nance-Horan syndrome 9 is needed to understand the possible correlation of molecular findings with the clinical phenotype. 
 
Figure 1.
 
The family pedigree showing segregation of Xp22 microsatellite markers. The disease haplotype is boxed. ?, undetermined allele.
Figure 1.
 
The family pedigree showing segregation of Xp22 microsatellite markers. The disease haplotype is boxed. ?, undetermined allele.
Table 1.
 
Clinical Features of the Affected Family Members
Table 1.
 
Clinical Features of the Affected Family Members
Subject Eye BSCVA Cataract Nystagmus Microcornea* Other Ocular Findings Dental Anomalies Anteverted Pinnae Brachymeta-Carpalia Cardiac Anomalies Dysmorphology
14 (Proband) OD 20/120 Aphakic + + Mild abnormalities
OS 20/400 Aphakic + + Esotropia
3 OD 20/300 Aphakic + + Exotropia Few teeth lost due to age Mild asymmetry
OS 20/120 Aphakic + +
5 OD 20/400 Aphakic + +
OS No PL + Pthysis
7 OD HMCF Aphakic + + Exotropia Midfacial retrusion and mandibular prognathism
OS 20/400 Aphakic + +
13 OD 20/20 Sutural
OS 20/20 Sutural
16 OD 20/30 Sutural coralliform; blue dot, PSC +
OS 20/30 +
20 OD 20/20 Sutural NA
OS 20/20 Sutural
24 OD 20/20 Blue dot + Low myopia NA
OS 20/40 Cortical + High myopia; amblyopia
27 OD 20/200 Cortical + High myopia; amblyopia NA
OS 20/20 +
28 OD 20/20 Blue dot Sutural
OS 20/20 Blue dot sutural
30 OD 20/20 Blue dot sutural
OS 20/20 Blue dot sutural
32 OD 20/20 + Mild abnormalities
OS 20/20 Cortical +
45 OD 20/20 Sutural + Low Myopia Mild facial asymmetry
OS 20/20 Sutural + Low Myopia
52 OD 20/40 PC IOL + Moderate abnormalities +
OS 20/120 PC IOL +
56 OD 20/160 PC IOL + + Amblyopic NA NA NA NA NA
OS 20/160 PC IOL + + Amblyopic
57 OD 20/30 Blue dot +
OS 20/20 Blue dot +
60 OD NA PC IOL + NA NA NA NA NA
OS NA PC IOL +
61 OD 20/40 Cortical + NA NA NA NA NA
OS 20/40 Cortical +
Figure 2.
 
(A) Slit lamp photograph of a carrier female (Fig. 1 , individual 24) showing a prominent white cortical cuneiform opacity in the inferior part of the lens, involving only the visual axis. Also visible were multiple spoke-like cortical opacities involving the lens periphery. (B) Anteverted pinnae in an affected male (Fig. 1 , individual 52). (C) Mild aberrations in tooth formation: notched, serrated teeth in a carrier female (Fig. 1 , individual 32). (D) Moderate aberrations in tooth formation and alignment: notched teeth and diastema in an affected male (Fig. 1 , individual 52). (E) Normal teeth in an affected male (Fig. 1 , individual 16) who had only cataract and microcornea. (F) Normal teeth in a normal female (Fig. 1 , individual 23).
Figure 2.
 
(A) Slit lamp photograph of a carrier female (Fig. 1 , individual 24) showing a prominent white cortical cuneiform opacity in the inferior part of the lens, involving only the visual axis. Also visible were multiple spoke-like cortical opacities involving the lens periphery. (B) Anteverted pinnae in an affected male (Fig. 1 , individual 52). (C) Mild aberrations in tooth formation: notched, serrated teeth in a carrier female (Fig. 1 , individual 32). (D) Moderate aberrations in tooth formation and alignment: notched teeth and diastema in an affected male (Fig. 1 , individual 52). (E) Normal teeth in an affected male (Fig. 1 , individual 16) who had only cataract and microcornea. (F) Normal teeth in a normal female (Fig. 1 , individual 23).
Figure 5.
 
Diagrammatic representation of the NHS gene and the identified mutations. fs, frameshift mutation.
Figure 5.
 
Diagrammatic representation of the NHS gene and the identified mutations. fs, frameshift mutation.
Table 2.
 
Results of Two-Point LOD Scores Assuming Recessive Transmission and Complete Penetrance
Table 2.
 
Results of Two-Point LOD Scores Assuming Recessive Transmission and Complete Penetrance
MARKER θ
0 0.001 0.01 0.05 0.1 0.2 0.3 0.4
DXS7104 −α −3.29 −1.33 −0.07 0.34 0.54 0.46 0.25
DXS987 2.11 2.10 2.07 1.93 1.74 1.33 0.87 0.36
DXS1195 2.11 2.10 2.07 1.93 1.74 1.33 0.87 0.36
DXS999 2.41 2.40 2.37 2.21 2.00 1.54 1.02 0.44
DXS7163 2.41 2.40 2.37 2.21 2.00 1.54 1.02 0.44
DXS1226 −α −3.29 −1.33 −0.07 0.34 0.54 0.46 0.25
Figure 3.
 
Sequence chromatogram showing the normal sequence (top) of exon 1 in control individual 23 and the truncating mutation, 115C→T, in exon 1 (bottom) in an affected male (individual 3), resulting in Q39X. Glutamine (CAG) to stop codon (TAG).
Figure 3.
 
Sequence chromatogram showing the normal sequence (top) of exon 1 in control individual 23 and the truncating mutation, 115C→T, in exon 1 (bottom) in an affected male (individual 3), resulting in Q39X. Glutamine (CAG) to stop codon (TAG).
Figure 4.
 
RT-PCR analysis of the NHS gene. (A) Expression of the ABL housekeeping gene (internal control) and its 296-bp product ensured that there was no genomic DNA contamination. (B) Expression of the NHS gene (501-bp region in exon 6). (C) Expression of the NHS gene’s major isoform (205-bp region in exon 1). Samples were from (lanes 1, 2, and 3) affected males, (lane 4) an unaffected related male, and (lane 5) an unaffected, unrelated male. Lane 6: molecular weight marker Phi X 174 DNA/Hinf digest (Bangalore Genei, Bangalore, India).
Figure 4.
 
RT-PCR analysis of the NHS gene. (A) Expression of the ABL housekeeping gene (internal control) and its 296-bp product ensured that there was no genomic DNA contamination. (B) Expression of the NHS gene (501-bp region in exon 6). (C) Expression of the NHS gene’s major isoform (205-bp region in exon 1). Samples were from (lanes 1, 2, and 3) affected males, (lane 4) an unaffected related male, and (lane 5) an unaffected, unrelated male. Lane 6: molecular weight marker Phi X 174 DNA/Hinf digest (Bangalore Genei, Bangalore, India).
The authors thank the family with Nance-Horan syndrome for their participation in the study and Hélène Dollfus (Clinique Ophthalmologique, Hopitaux Universitaires de Strasbourg, Strasbourg, France) for help with the dysmorphology. 
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Figure 1.
 
The family pedigree showing segregation of Xp22 microsatellite markers. The disease haplotype is boxed. ?, undetermined allele.
Figure 1.
 
The family pedigree showing segregation of Xp22 microsatellite markers. The disease haplotype is boxed. ?, undetermined allele.
Figure 2.
 
(A) Slit lamp photograph of a carrier female (Fig. 1 , individual 24) showing a prominent white cortical cuneiform opacity in the inferior part of the lens, involving only the visual axis. Also visible were multiple spoke-like cortical opacities involving the lens periphery. (B) Anteverted pinnae in an affected male (Fig. 1 , individual 52). (C) Mild aberrations in tooth formation: notched, serrated teeth in a carrier female (Fig. 1 , individual 32). (D) Moderate aberrations in tooth formation and alignment: notched teeth and diastema in an affected male (Fig. 1 , individual 52). (E) Normal teeth in an affected male (Fig. 1 , individual 16) who had only cataract and microcornea. (F) Normal teeth in a normal female (Fig. 1 , individual 23).
Figure 2.
 
(A) Slit lamp photograph of a carrier female (Fig. 1 , individual 24) showing a prominent white cortical cuneiform opacity in the inferior part of the lens, involving only the visual axis. Also visible were multiple spoke-like cortical opacities involving the lens periphery. (B) Anteverted pinnae in an affected male (Fig. 1 , individual 52). (C) Mild aberrations in tooth formation: notched, serrated teeth in a carrier female (Fig. 1 , individual 32). (D) Moderate aberrations in tooth formation and alignment: notched teeth and diastema in an affected male (Fig. 1 , individual 52). (E) Normal teeth in an affected male (Fig. 1 , individual 16) who had only cataract and microcornea. (F) Normal teeth in a normal female (Fig. 1 , individual 23).
Figure 5.
 
Diagrammatic representation of the NHS gene and the identified mutations. fs, frameshift mutation.
Figure 5.
 
Diagrammatic representation of the NHS gene and the identified mutations. fs, frameshift mutation.
Figure 3.
 
Sequence chromatogram showing the normal sequence (top) of exon 1 in control individual 23 and the truncating mutation, 115C→T, in exon 1 (bottom) in an affected male (individual 3), resulting in Q39X. Glutamine (CAG) to stop codon (TAG).
Figure 3.
 
Sequence chromatogram showing the normal sequence (top) of exon 1 in control individual 23 and the truncating mutation, 115C→T, in exon 1 (bottom) in an affected male (individual 3), resulting in Q39X. Glutamine (CAG) to stop codon (TAG).
Figure 4.
 
RT-PCR analysis of the NHS gene. (A) Expression of the ABL housekeeping gene (internal control) and its 296-bp product ensured that there was no genomic DNA contamination. (B) Expression of the NHS gene (501-bp region in exon 6). (C) Expression of the NHS gene’s major isoform (205-bp region in exon 1). Samples were from (lanes 1, 2, and 3) affected males, (lane 4) an unaffected related male, and (lane 5) an unaffected, unrelated male. Lane 6: molecular weight marker Phi X 174 DNA/Hinf digest (Bangalore Genei, Bangalore, India).
Figure 4.
 
RT-PCR analysis of the NHS gene. (A) Expression of the ABL housekeeping gene (internal control) and its 296-bp product ensured that there was no genomic DNA contamination. (B) Expression of the NHS gene (501-bp region in exon 6). (C) Expression of the NHS gene’s major isoform (205-bp region in exon 1). Samples were from (lanes 1, 2, and 3) affected males, (lane 4) an unaffected related male, and (lane 5) an unaffected, unrelated male. Lane 6: molecular weight marker Phi X 174 DNA/Hinf digest (Bangalore Genei, Bangalore, India).
Table 1.
 
Clinical Features of the Affected Family Members
Table 1.
 
Clinical Features of the Affected Family Members
Subject Eye BSCVA Cataract Nystagmus Microcornea* Other Ocular Findings Dental Anomalies Anteverted Pinnae Brachymeta-Carpalia Cardiac Anomalies Dysmorphology
14 (Proband) OD 20/120 Aphakic + + Mild abnormalities
OS 20/400 Aphakic + + Esotropia
3 OD 20/300 Aphakic + + Exotropia Few teeth lost due to age Mild asymmetry
OS 20/120 Aphakic + +
5 OD 20/400 Aphakic + +
OS No PL + Pthysis
7 OD HMCF Aphakic + + Exotropia Midfacial retrusion and mandibular prognathism
OS 20/400 Aphakic + +
13 OD 20/20 Sutural
OS 20/20 Sutural
16 OD 20/30 Sutural coralliform; blue dot, PSC +
OS 20/30 +
20 OD 20/20 Sutural NA
OS 20/20 Sutural
24 OD 20/20 Blue dot + Low myopia NA
OS 20/40 Cortical + High myopia; amblyopia
27 OD 20/200 Cortical + High myopia; amblyopia NA
OS 20/20 +
28 OD 20/20 Blue dot Sutural
OS 20/20 Blue dot sutural
30 OD 20/20 Blue dot sutural
OS 20/20 Blue dot sutural
32 OD 20/20 + Mild abnormalities
OS 20/20 Cortical +
45 OD 20/20 Sutural + Low Myopia Mild facial asymmetry
OS 20/20 Sutural + Low Myopia
52 OD 20/40 PC IOL + Moderate abnormalities +
OS 20/120 PC IOL +
56 OD 20/160 PC IOL + + Amblyopic NA NA NA NA NA
OS 20/160 PC IOL + + Amblyopic
57 OD 20/30 Blue dot +
OS 20/20 Blue dot +
60 OD NA PC IOL + NA NA NA NA NA
OS NA PC IOL +
61 OD 20/40 Cortical + NA NA NA NA NA
OS 20/40 Cortical +
Table 2.
 
Results of Two-Point LOD Scores Assuming Recessive Transmission and Complete Penetrance
Table 2.
 
Results of Two-Point LOD Scores Assuming Recessive Transmission and Complete Penetrance
MARKER θ
0 0.001 0.01 0.05 0.1 0.2 0.3 0.4
DXS7104 −α −3.29 −1.33 −0.07 0.34 0.54 0.46 0.25
DXS987 2.11 2.10 2.07 1.93 1.74 1.33 0.87 0.36
DXS1195 2.11 2.10 2.07 1.93 1.74 1.33 0.87 0.36
DXS999 2.41 2.40 2.37 2.21 2.00 1.54 1.02 0.44
DXS7163 2.41 2.40 2.37 2.21 2.00 1.54 1.02 0.44
DXS1226 −α −3.29 −1.33 −0.07 0.34 0.54 0.46 0.25
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