A novel locus (RP24) for X-linked retinitis pigmentosa maps to Xq26-27.

Two genetic loci, RP2 and RP3, for X-linked retinitis pigmentosa (XLRP) have been localized to Xp11.3-11.23 and Xp21.1, respectively. RP3 appears to account for 70% of XLRP families; however, mutations in the RPGR gene (isolated from the RP3 region) are identified in only 20% of affected families. Close location of XLRP loci at Xp and a lack of unambiguous clinical criteria do not permit assignment of genetic subtype in a majority of XLRP families; nonetheless, in some pedigrees, both RP2 and RP3 could be excluded as the causative locus. We report the mapping of a novel locus, RP24, by haplotype and linkage analysis of a single XLRP pedigree. The RP24 locus was identified at Xq26-27 by genotyping 52 microsatellite markers spanning the entire X chromosome. A maximum LOD score of 4.21 was obtained with DXS8106. Haplotype analysis assigned RP24 within a 23-cM region between the DXS8094 (proximal) and DXS8043 (distal) markers. Other chromosomal regions and known XLRP loci were excluded by obligate recombination events between markers in those regions and the disease locus. Hemizygotes from the RP24 family have early onset of rod photoreceptor dysfunction; cone receptor function is normal at first, but there is progressive loss. Patients at advanced stages show little or no detectable rod or cone function and have clinical hallmarks of typical RP. Mapping of the RP24 locus expands our understanding of the genetic heterogeneity in XLRP and will assist in development of better tools for diagnosis.     

Identification of novel RPGR (retinitis pigmentosa GTPase regulator) mutations in a subset of X-linked retinitis pigmentosa families segregating with the RP3 locus

The X-linked form of retinitis pigmentosa (XLRP) is a severe disease of the retina, characterised by night blindness and visual field constriction in a degenerative process, culminating with complete loss of sight within the third decade of life. Genetic mapping studies have identified two major loci for XLRP: RP3 (70%–75% of XLRP) and RP2 (20%–25% of XLRP). The RPGR (retinitis pigmentosa GTPase regulator) gene has been cloned within the RP3 genomic interval and it has been shown that 10%–20% of XLRP families have mutations in this gene. Here, we describe a single-strand conformational polymorphism-based mutation screening of RPGR in a pool of 29 XLRP families for which the disease segregates with the RP3 locus, in order to investigate the proportion of RP3 families with RPGR mutations and to relate the results to previous reports. Five different new mutations have been identified: two splice site mutations for exon 1 and three frameshift mutations in exons 7, 10 and 11. The percentage of RPGR mutations identified is 17% (5/29) in our genetically well-defined population. This figure is comparable to the percentage of RP2 gene mutations that we have detected in our entire XLRP patient pool (10%–15%). A correlation of RPGR mutations with phenotype in the families described in this study and the biochemical characterisation of reported mutations may provide insights into the function of the protein. Electronic Publication     

A recombination outside the BB deletion refines the location of the X linked retinitis pigmentosa locus RP3.

Genetic loci for X-linked retinitis pigmentosa (XLRP) have been mapped between Xp11.22 and Xp22.13 (RP2, RP3, RP6, and RP15). The RP3 gene, which is responsible for the predominant form of XLRP in most Caucasian populations, has been localized to Xp21.1 by linkage analysis and the map positions of chromosomal deletions associated with the disease. Previous linkage studies have suggested that RP3 is flanked by the markers DXS1110 (distal) and OTC (proximal). Patient BB was thought to have RP because of a lesion at the RP3 locus, in addition to chronic granulomatous disease, Duchenne muscular dystrophy (DMD), mild mental retardation, and the McLeod phenotype. This patient carried a deletion extending approximately 3 Mb from DMD in Xp21.3 to Xp21.1, with the proximal breakpoint located approximately 40 kb centromeric to DXS1110. The RP3 gene, therefore, is believed to reside between DXS1110 and the proximal breakpoint of the BB deletion. In order to refine the location of RP3 and to ascertain patients with RP3, we have been analyzing several XLRP families for linkage to Xp markers. Linkage analysis in an American family of 27 individuals demonstrates segregation of XLRP with markers in Xp21.1, consistent with the RP3 subtype. One affected mate shows a recombination event proximal to DXS1110. Additional markers within the DXS1110-OTC interval show that the crossover is between two novel polymorphic markers, DXS8349 and M6, both of which are present in BB DNA and lie centromeric to the proximal breakpoint. This recombination places the XLRP mutation in this family outside the BB deletion and redefines the location of RP3.     

Remapping of the RP15 locus for X-linked cone-rod degeneration to Xp11.4-p21.1, and identification of a de novo insertion in the RPGR exon ORF15

X-linked forms of retinitis pigmentosa (XLRP) are among the most severe, because of their early onset, often leading to significant vision loss before the 4th decade. Previously, the RP15 locus was assigned to Xp22, by linkage analysis of a single pedigree with "X-linked dominant cone-rod degeneration." After clinical reevaluation of a female in this pedigree identified her as affected, we remapped the disease to a 19.5-cM interval (DXS1219-DXS993) at Xp11.4-p21.1. This new interval overlapped both RP3 (RPGR) and COD1. Sequencing of the previously published exons of RPGR revealed no mutations, but a de novo insertion was detected in the new RPGR exon, ORF15. The identification of an RPGR mutation in a family with a severe form of cone and rod degeneration suggests that RPGR mutations may encompass a broader phenotypic spectrum than has previously been recognized in "typical" retinitis pigmentosa.     

A comprehensive mutation analysis of RP2 and RPGR in a North American cohort of families with X-linked retinitis pigmentosa

X-linked retinitis pigmentosa (XLRP) is a clinically and genetically heterogeneous degenerative disease of the retina. At least five loci have been mapped for XLRP; of these, RP2 and RP3 account for 10%-20% and 70%-90% of genetically identifiable disease, respectively. However, mutations in the respective genes, RP2 and RPGR, were detected in only 10% and 20% of families with XLRP. Mutations in an alternatively spliced RPGR exon, ORF15, have recently been shown to account for 60% of XLRP in a European cohort of 47 families. We have performed, in a North American cohort of 234 families with RP, a comprehensive screen of the RP2 and RPGR (including ORF15) genes and their 5' upstream regions. Of these families, 91 (39%) show definitive X-linked inheritance, an additional 88 (38%) reveal a pattern consistent with X-linked disease, and the remaining 55 (23%) are simplex male patients with RP who had an early onset and/or severe disease. In agreement with the previous studies, we show that mutations in the RP2 gene and in the original 19 RPGR exons are detected in <10% and approximately 20% of XLRP probands, respectively. Our studies have revealed RPGR-ORF15 mutations in an additional 30% of 91 well-documented families with X-linked recessive inheritance and in 22% of the total 234 probands analyzed. We suggest that mutations in an as-yet-uncharacterized RPGR exon(s), intronic changes, or another gene in the region might be responsible for the disease in the remainder of this North American cohort. We also discuss the implications of our studies for genetic diagnosis, genotype-phenotype correlations, and gene-based therapy.     

Molecular analysis in Brazilian cystic fibrosis patients reveals five novel mutations

We have performed molecular genetic analyses on 160 Brazilian patients diagnosed with cystic fibrosis (CF). Screening of mutations in 320 CF chromosomes was performed through single strand conformation polymorphism (SSCP) and heteroduplex analyses assay followed by DNA sequencing of the 27 exons and exon/intron boundaries of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The frequency of CFTR variants of T-tract length of intron 8 (IVS8 Tn) was also investigated. This analysis enabled the detection of 232/320 CF mutations (72.2%) and complete genotyping of 61% of the patients. The deltaF508 mutation was found in 48.4% of the alleles. Another fifteen mutations (previously reported) were detected: G542X, R1162X, N1303K, R334W, W1282X, G58E, L206W, R553X, 621+1G-->T, V232D, 1717-1G-->A, 2347 delG, R851L, 2789+5G-->A, and W1089X. Five novel mutations were identified, V201M (exon 6a), Y275X (exon 6b), 2686 insT (exon 14a), 3171 delC (exon 17a), and 3617 delGA (exon 19). These results contribute to the molecular characterization of CF in the Brazilian population. In addition, the identification of the novel mutation Y275X allowed prenatal diagnosis in a high-risk fetus.     

A family with RP3 type of X-linked retinitis pigmentosa: an association with ciliary abnormalities

Summary The results of linkage analysis in a family with X-linked retinitis pigmentosa (XLRP) are presented. Probe M27B (DXS255), localised to Xp11.22, was only loosely linked to XLRP, whereas pHOC3 (OTC), in the more distal Xp21.1 region, was tightly linked. In this family, the conditional probability of an RP3 locus (in Xp21.1–p11.4) was found to be 0.978 compared with 0.021 for an RP2 locus (in Xp11.4–p11.2). Risk assessment showed that 2 out of 4 at risk females showing no clinical abnormality have a high probability of being genetic carriers of XLRP. Some affected males have recurrent respiratory infections as a result of a condition indistinguishable from the immotile cilia syndrome; indeed, there is an association between XLRP and susceptibility to respiratory infections in the majority of affected males. The possibility that previously observed ciliary abnormalities in XLRP patients might be associated specifically with an RP3 locus abnormality is discussed.     

Physical mapping at a potential X-linked retinitis pigmentosa locus (RP3) by pulsed-field gel electrophoresis.

A genetic locus (RP3) for X-linked retinitis pigmentosa (XLRP) has been assigned to Xp21 by genetic linkage studies and has been supported by two Xp21 male deletion patients with XLRP. RP3 appears to be the most centromeric of several positioned loci, including chronic granulomatous disease (CGD), McLeod phenotype (XK), and Duchenne muscular dystrophy (DMD). In one patient, BB, the X-chromosome deletion includes RP3 and extends to within the DMD locus. Using a DMD cDNA, the centromeric endpoint of this patient was cloned and used as a starting point for chromosome walking along a normal X chromosome. A single-copy probe, XH1.4, positioned near the centromeric junction but deleted in BB, was used along with a CGD cDNA probe to establish a refined long-range physical map. Both probes recognized a common SfiI fragment of 205 kb. As the CGD gene covers approximately 30-60 kb, the RP3 locus has been restricted to approximately 150-170 kb. A CpG island, potentially marking a new gene, was identified within the SfiI fragment at a position approximately 35 kb from the deletion endpoint in BB.     

Localization of the microsatellite probe DXS426 between DXS7 and DXS255 on Xp and linkage to X-linked retinitis pigmentosa.

The microsatellite marker DXS426 maps to the interval Xp21.1-Xp11.21, the chromosomal region which contains two loci for X-linked retinitis pigmentosa (XLRP; RP2 and RP3). We have refined the localization of DXS426 both physically, by mapping it to a deletion which spans the interval Xp21.3-Xp11.23, and genetically, by studying multiply informative crossovers which indicate that DXS426 lies between DXS7 and DXS255 (i.e., Xp11.4-Xp11.22). As this is the region which contains the RP2 gene, RP2 families could be identified on the basis of linkage of XLRP to DXS426. Multiply informative crossovers in two RP2 families indicate that the most likely location of the RP2 gene is between DXS426 and DXS7. DXS426 is therefore an important highly informative marker for the purposes of carrier detection and early diagnosis of RP2 and for the localization of the disease gene.     

RP2 and RPGR mutations and clinical correlations in patients with X-linked retinitis pigmentosa

We determined the mutation spectrum of the RP2 and RPGR genes in patients with X-linked retinitis pigmentosa (XLRP) and searched for correlations between categories of mutation and severity of disease. We screened 187 unrelated male patients for mutations, including 135 with a prior clinical diagnosis of XLRP, 11 with probable XLRP, 30 isolate cases suspected of having XLRP, and 11 with cone-rod degeneration. Mutation screening was performed by single-strand conformation analysis and by sequencing of all RP2 exons and RPGR exons 1-14, ORF15, and 15a. The refractive error, visual acuity, final dark-adapted threshold, visual field area, and 30-Hz cone electroretinogram (ERG) amplitude were measured in each patient. Among the 187 patients, we found 10 mutations in RP2, 2 of which are novel, and 80 mutations in RPGR, 41 of which are novel; 66% of the RPGR mutations were within ORF15. Among the 135 with a prior clinical diagnosis of XLRP, mutations in the RP2 and RPGR genes were found in 9 of 135 (6.7%) and 98 of 135 (72.6%), respectively, for a total of 79% of patients. Patients with RP2 mutations had, on average, lower visual acuity but similar visual field area, final dark-adapted threshold, and 30-Hz ERG amplitude compared with those with RPGR mutations. Among patients with RPGR mutations, those with ORF15 mutations had, on average, a significantly larger visual field area and a borderline larger ERG amplitude than did patients with RPGR mutations in exons 1-14. Among patients with ORF15 mutations, regression analyses showed that the final dark-adapted threshold became lower (i.e., closer to normal) and that the 30-Hz ERG amplitude increased as the length of the wild-type ORF15 amino acid sequence increased. Furthermore, as the length of the abnormal amino acid sequence following ORF15 frameshift mutations increased, the severity of disease increased.     

Novel Mutations of RPGR in Chinese Retinitis Pigmentosa Patients and the Genotype-Phenotype Correlation

X-linked Retinitis Pigmentosa (XLRP) accounts for 10–20% of all RP cases, and represents the most severe subtype of this disease. Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) gene are the most common causes of XLRP, accounting for over 70–75% of all XLRP cases. In this work, we analyzed all the exons of RPGR gene with Sanger sequencing in seven Chinese XLRP families, two of these with a provisional diagnosis of adRP but without male-to-male transmission. Three novel deletions (c.2233_34delAG; c.2236_37delGA and c.2403_04delAG) and two known nonsense mutations (c.851C→G and c.2260G→T) were identified in five families. Two novel deletions (c.2233_34delAG and c.2236_37delGA) resulted in the same frame shift (p.E746RfsX22), created similar phenotype in Family 3 and 4. The novel deletion (c.2403_04delAG; p.E802GfsX31) resulted in both XLRP and x-linked cone-rod dystrophy within the male patients of family 5, which suggested the presence of either genetic or environmental modifiers, or both, play a substantial role in disease expression. Genotype-phenotype correlation analysis suggested that (1) both patients and female carriers with mutation in Exon 8 (Family 1) manifest more severe disease than did those with ORF15 mutations (Family 2&3&4); (2) mutation close to downstream of ORF15 (Family 5) demonstrate the early preferential loss of cone function with moderate loss of rod function.     

Mutations of the phenylalanine hydroxylase gene in patients with phenylketonuria in Shanxi,China

The variation in mutations in exons 3, 6, 7, 11 and 12 of the phenylalanine hydroxylase (PAH) gene was investigated in 59 children with phenylketonuria (PKU) and 100 normal children. Three single nucleotide polymorphisms were detected by sequence analysis. The mutational frequencies of cDNA 696, cDNA 735 and cDNA 1155 in patients were 96.2%, 76.1% and 7.6%, respectively, whereas in healthy children the corresponding frequencies were 97.0%, 77.3% and 8.3%. In addition, 81 mutations accounted for 61.0% of the mutant alleles. R111X, H64 > TfsX9 and S70 del accounted for 5.1%, 0.8% and 0.8% mutation of alleles in exon 3, whereas EX6-96A > G accounted for 10.2% mutation of alleles in exon 6. R243Q had the highest incidence in exon 7 (12.7%), followed by Ivs7 + 2 T > A (5.1%) and T278I (2.5%). G247V, R252Q, L255S, R261Q and E280K accounted for 0.8% while Y356X and V399V accounted for 5.9% and 5.1%, respectively, in exon 11. R413P and A434D accounted for 5.9% and 2.5%, respectively, in exon 12. Seventy-two variant alleles accounted for the 16 mutations observed here. The mutation characteristics and distributions demonstrated that EX6-96A > G and R243Q were the hot regions for mutations in the PAH gene in Shanxi patients with PKU.     

AG-dependent 3'-splice sites are predisposed to aberrant splicing due to a mutation at the first nucleotide of an exon

In pre-mRNA splicing, a conserved AG/G at the 3'-splice site is recognized by U2AF(35). A disease-causing mutation abrogating the G nucleotide at the first position of an exon (E(+1)) causes exon skipping in GH1, FECH and EYA1, but not in LPL or HEXA. Knockdown of U2AF(35) enhanced exon skipping in GH1 and FECH. RNA-EMSA revealed that wild-type FECH requires U2AF(35) but wild-type LPL does not. A series of artificial mutations in the polypyrimidine tracts of GH1, FECH, EYA1, LPL and HEXA disclosed that a stretch of at least 10-15 pyrimidines is required to ensure normal splicing in the presence of a mutation at E(+1). Analysis of nine other disease-causing mutations at E(+1) detected five splicing mutations. Our studies suggest that a mutation at the AG-dependent 3'-splice site that requires U2AF(35) for spliceosome assembly causes exon skipping, whereas one at the AG-independent 3'-splice site that does not require U2AF(35) gives rise to normal splicing. The AG-dependence of the 3'-splice site that we analyzed in disease-causing mutations at E(+1) potentially helps identify yet unrecognized splicing mutations at E(+1).     

Molecular defects in the α-N-acetylglucosaminidase gene in Italian Sanfilippo type B patients

Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is a rare autosomal recessive disorder characterized by the inability to degrade heparan sulfate because of a deficiency of the lysosomal enzyme alpha-N-acetylglucosaminidase (NAGLU). We performed mutation screening in a group of 20 patients, identyifing 28 mutations, 14 of which were novel (L35F, 204delC, 221insGCGCG, G82D, W156C, 507delC, IVS3+1G-->A, E336X, V501G, R520W, S534Y, W649C, 1953insGCCA, 2185delAGA). Four of these mutations were found in homozygosity and only one was seen in two different patients, showing the remarkable molecular heterogeneity of the disease. Mutation IVS3+1G-->A produces aberrant RNA splicing: it represents a base substitution from G to A of the invariant GT dinucleotides at the splicing donor site of intron 3 resulting in the skipping of exon 3 and both exons 2 and 3. Transient transfection of COS cells, by DNA mutagenized with NAGLU mutations, produced enzymatic molecules without activity, demonstrating the deleterious nature of the defects. Metabolic labeling of transfected mutants suggested a normal synthesis of the involved polypeptide for missense alterations, whereas increased protein or mRNA instability was shown for nonsense and most of the frameshift mutations.     

Heterogeneous mutations in the glycogen-debranching enzyme gene are responsible for glycogen storage disease type IIIa in Japan

Glycogen storage disease type IIIa (GSD IIIa) is an autosomal recessive disorder caused by deficiency of the glycogen-debranching enzyme (AGL). Recent studies of the AGL gene have revealed the prevalent mutations in North African Jewish and Caucasian populations, but whether these common mutations are present in other ethnic groups remains unclear. We have investigated eight Japanese GSD IIIa patients from seven families and identified seven mutations, including one splicing mutation (IVS 14+1G-->T) previously reported by us, together with six novel ones: a nonsense mutation (L124X), a splice site mutation (IVS29-1G-->C), a 1-bp deletion (587delC), a 2-bp deletion (4216-4217delAG), a 1-bp insertion (2072-2073insA), and a 3-bp insertion (4735-4736insTAT). The last mutation results in insertion of a tyrosine residue at a putative glycogen-binding site, and the rest are predicted to cause synthesis of truncated proteins lacking the glycogen-binding site at the carboxyl terminal. Thirteen novel polymorphisms have also been revealed in this study: three amino acid substitutions (R387Q, G1115R, and E1343 K), one silent point mutation (L298L), one nucleotide change in the 5'-noncoding region, and eight nucleotide changes in introns. Haplotype analysis with combinations of these polymorphic markers showed L124X, IVS14+1G-->T, and 4216-4217delAG to be on different haplotypes. These results demonstrate the importance of the integrity of the carboxy terminal domain in the AGL protein and the molecular heterogeneity of GSD IIIa in Japan.     

In vitro replication of primer-templates containing benzo[a]pyrene adducts by exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment): effect of sequence context on lesion bypass

The presence of benzo[a]pyrene diol epoxide (B[a]PDE) adducts in DNA is known to interfere with DNA replication. Kinetic studies of nucleotide insertion by exonuclease-deficient E. coli DNA polymerase I (Klenow fragment) across from either the (+)-trans- or the (+)-cis-B[a]P-N(2)-dG adduct in the 5'-CGT-3' sequence context indicated that the rate of nucleotide incorporation followed the order: dAMP > dGMP > dTMP > dCMP, which did not correlate with the mutational spectrum observed for these adducts in this sequence in E. coli (mostly G-->A transitions). Interestingly, a kinetic analysis of extension past the adduct showed that, unlike other sequences studied, the primer-template was extended best when dT was positioned at the 3'-terminus of the primer across from either a (+)-trans- or a (+)-cis-B[a]P-N(2)-dG adduct. In contrast, when the (+)-trans-B[a]P-N(2)-dG adduct was positioned in the 5'-TGC-3' sequence context, which gives predominantly G-->T mutations in E. coli, extension was detectable only when dA was positioned across from the adduct. These data provide the first in vitro evidence that may explain why G-->A transitions, rather than the G-->T transversions found in other sequences, are preferred in the 5'-CGT-3' sequence in vivo.     

Heterogeneity Analysis in 40 X-linked Retinitis Pigmentosa Families

Analysis of genetic heterogeneity in 40 kindreds with X-linked retinitis pigmentosa (XLRP), with 20 polymorphic markers, showed that significant heterogeneity is present (P=.001) and that 56% of kindreds are of RP3 type and that 26% are of RP2 type. The location of the RP3 locus was found to be 0.4 cM distal to OTC in the Xp21.1 region, and that of the RP2 locus was 6.5 cM proximal to DXS7 in Xp11.2-p11.3. Bayesian probabilities of linkage to RP2, RP3, or to neither locus were calculated. This showed that 20 of 40 kindreds could be assigned to one or the other locus, with a probability >.70 (14 kindreds with RP3 and 6 kindreds with RP2 disease). A further three kindreds were found to be unlinked to either locus, with a probability >.8. The remaining 17 kindreds could not be classified unambiguously. This highlights the difficulty of classifying families in the presence of genetic heterogeneity, where the two loci are separated by an estimated 16 cM.