Glucocerebrosidase mutations in Gaucher disease.

BACKGROUND: Thirty-six mutations that cause Gaucher disease, the most common glycolipid storage disorder, are known. Although both alleles of most patients with the disease contain one of these mutations, in a few patients one or both disease-producing alleles have remained unidentified. Identification of mutations in these patients is useful for genetic counseling. MATERIALS AND METHODS: The DNA from 23 Gaucher disease patients in whom at least one glucocerebrosidase allele did not contain any of the 36 previously described mutations has been examined by single strand conformation polymorphism (SSCP) analysis, followed by sequencing of regions in which abnormalities were detected. RESULTS: Eight previously undescribed mutations were detected. In exon 3, a deletion of a cytosine at cDNA nt 203 was found. In exon 6, three missense mutations were identified: a C-->A transversion at cDNA nt 644 (Ala176-->Asp), a C-->A transversion at cDNA nt 661 that resulted in a (Pro182-->Thr), and a G-->A transition at cDNA nt 721 (Gly202-->Arg). Two missense mutations were found in exon 7: a G-->A transition at cDNA nt 887 (Arg257-->Gln) and a C-->T at cDNA nt 970 (Arg285-->Cys). Two missense mutations were found in exon 9: a T-->G at cDNA nt 1249 (Trp378-->Gly) and a G-->A at cDNA nt 1255 (Asp380-->Asn). In addition to these disease-producing mutations, a silent C-->G transversion at cDNA nt 1431, occurring in a gene that already contained the 1226G mutation, was found in one family. CONCLUSIONS: The mutations described here and previously known can be classified as mild, severe, or lethal, on the basis of their effect on enzyme production and on clinical phenotype, and as polymorphic or sporadic, on the basis of the haplotype in which they are found. Rare mutations such as the new ones described here are sporadic in nature.     

Genetic homogeneity of Pelizaeus-Merzbacher disease: tight linkage to the proteolipoprotein locus in 16 affected families. PMD Clinical Group.

Among the numerous leukodystrophies that have an early onset and no biochemical markers, Pelizaeus-Merzbacher disease (PMD) is one that can be identified using strict clinical criteria and demonstrating an abnormal formation of myelin that is restricted to the CNS in electrophysiological studies and brain magnetic resonance imaging (MRI). In PMD, 12 different base substitutions and one total deletion of the genomic region containing the PLP gene have been reported, but, despite extensive analysis, PLP exon mutations have been found in only 10%-25% of the families analyzed. To test the genetic homogeneity of this disease, we have carried out linkage analysis with polymorphic markers of the PLP genomic region in 16 families selected on strict diagnostic criteria of PMD. We observed a tight linkage of the PMD locus with markers of the PLP gene (cDNA PLP, exon IV polymorphism) and of the Xq22 region (DXS17, DXS94, and DXS287), whereas the markers located more proximally (DXYS1X and DXS3) or distally (DXS11) were not linked to the PMD locus. Multipoint analysis gave a maximal location score for the PMD locus (13.98) and the PLP gene (8.32) in the same interval between DXS94 and DXS287, suggesting that in all families PMD is linked to the PLP locus. Mutations of the extraexonic PLP gene sequences or of another unknown close gene could be involved in PMD. In an attempt to identify molecular defects of this genomic region that are responsible for PMD, these results meant that RFLP analysis could be used to improve genetic counseling for the numerous affected families in which a PLP exon mutation could not be demonstrated.     

SNP Linkage Analysis and Whole Exome Sequencing Identify a Novel POU4F3 Mutation in Autosomal Dominant Late-Onset Nonsyndromic Hearing Loss (DFNA15)

Autosomal dominant non-syndromic hearing loss (AD-NSHL) is one of the most common genetic diseases in human and is well-known for the considerable genetic heterogeneity. In this study, we utilized whole exome sequencing (WES) and linkage analysis for direct genetic diagnosis in AD-NSHL. The Korean family had typical AD-NSHL running over 6 generations. Linkage analysis was performed by using genome-wide single nucleotide polymorphism (SNP) chip and pinpointed a genomic region on 5q31 with a significant linkage signal. Sequential filtering of variants obtained from WES, application of the linkage region, bioinformatic analyses, and Sanger sequencing validation identified a novel missense mutation Arg326Lys (c.977G>A) in the POU homeodomain of the POU4F3 gene as the candidate disease-causing mutation in the family. POU4F3 is a known disease gene causing AD-HSLH (DFNA15) described in 5 unrelated families until now each with a unique mutation. Arg326Lys was the first missense mutation affecting the 3^rd alpha helix of the POU homeodomain harboring a bipartite nuclear localization signal sequence. The phenotype findings in our family further supported previously noted intrafamilial and interfamilial variability of DFNA15. This study demonstrated that WES in combination with linkage analysis utilizing bi-allelic SNP markers successfully identified the disease locus and causative mutation in AD-NSHL.     

Allelic heterogeneity at the equine KIT locus in dominant white (W) horses

White coat color has been a highly valued trait in horses for at least 2,000 years. Dominant white (W) is one of several known depigmentation phenotypes in horses. It shows considerable phenotypic variation, ranging from ~50% depigmented areas up to a completely white coat. In the horse, the four depigmentation phenotypes roan, sabino, tobiano, and dominant white were independently mapped to a chromosomal region on ECA 3 harboring the KIT gene. KIT plays an important role in melanoblast survival during embryonic development. We determined the sequence and genomic organization of the ~82 kb equine KIT gene. A mutation analysis of all 21 KIT exons in white Franches-Montagnes Horses revealed a nonsense mutation in exon 15 (c.2151C>G, p.Y717X). We analyzed the KIT exons in horses characterized as dominant white from other populations and found three additional candidate causative mutations. Three almost completely white Arabians carried a different nonsense mutation in exon 4 (c.706A>T, p.K236X). Six Camarillo White Horses had a missense mutation in exon 12 (c.1805C>T, p.A602V), and five white Thoroughbreds had yet another missense mutation in exon 13 (c.1960G>A, p.G654R). Our results indicate that the dominant white color in Franches-Montagnes Horses is caused by a nonsense mutation in the KIT gene and that multiple independent mutations within this gene appear to be responsible for dominant white in several other modern horse populations.     

Exome Sequencing Identifies a Mutation in EYA4 as a Novel Cause of Autosomal Dominant Non-Syndromic Hearing Loss

Autosomal dominant non-syndromic hearing loss is highly heterogeneous, and eyes absent 4 (EYA4) is a disease-causing gene. Most EYA4 mutations founded in the Eya-homologous region, however, no deafness causative missense mutation in variable region of EYA4 have previously been found. In this study, we identified a pathogenic missense mutation located in the variable region of the EYA4 gene for the first time in a four-generation Chinese family with 57 members. Whole-exome sequencing (WES) was performed on samples from one unaffected and two affected individuals to systematically search for deafness susceptibility genes, and the candidate mutations and the co-segregation of the phenotype were verified by polymerase chain reaction amplification and by Sanger sequencing in all of the family members. Then, we identified a novel EYA4 mutation in exon 8, c.511G>C; p.G171R, which segregated with postlingual and progressive autosomal dominant sensorineural hearing loss (SNHL). This report is the first to describe a missense mutation in the variable region domain of the EYA4 gene, which is not highly conserved in many species, indicating that the potential unconserved role of 171G>R in human EYA4 function is extremely important.     

A (G-to-A) mutation in the initiation codon of the proteolipid protein gene causing a relatively mild form of Pelizaeus-Merzbacher disease in a Dutch family

Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive disorder that is characterized by dysmyelination of the central nervous system resulting from mutations in the proteolipid protein (PLP) gene. Mutations causing either overexpression or expression of a truncated form of PLP result in oligodendrocyte cell death because of accumulation of PLP in the endoplasmic reticulum. It has therefore been hypothesized that absence of the protein should result in a less severe phenotype. However, until now, only one patient has been described with a complete deletion of the PLP gene. We report a Dutch family with a relatively mild form of PMD, in which the disease cosegregates with a (G-to-A) mutation in the initiation codon of the PLP gene. This mutation should cause the total absence of PLP and is therefore in agreement with the hypothesis that absence of PLP leads to a mild form of PMD.     

Progressive ataxia due to a missense mutation in a calcium-channel gene.

We describe a family with severe progressive cerebellar ataxia involving the trunk, the extremities, and speech. The proband, who has prominent atrophy of the cerebellum, shown by magnetic resonance imaging, was confined to a wheelchair at the age of 44 years. Two sons have episodes of vertigo and ataxia that are not responsive to acetazolamide. Quantitative eye-movement testing showed a consistent pattern of abnormalities localizing to the cerebellum. Genotyping suggested linkage to chromosome 19p, and SSCP showed an aberrant migrating fragment in exon 6 of the calcium-channel gene CACNA1A, which cosegregated with the disease. Sequencing of exon 6 identified a G-->A transposition in one allele, at nucleotide 1152, resulting in a predicted glycine-to-arginine substitution at codon 293. The CAG-repeat expansion associated with spinocerebellar ataxia 6 was not present in any family members. This family is unique in having a non-CAG-repeat mutation that leads to severe progressive ataxia. Since a great deal is known about the function of calcium channels, we speculate on how this missense mutation leads to the combination of clinical symptoms and signs.     

Identification of genetic mutations in Japanese patients with fructose-1,6-bisphosphatase deficiency.

Fructose-1,6-bisphosphatase (FBPase) deficiency is an autosomal recessive inherited disorder and may cause sudden unexpected infant death. We reported the first case of molecular diagnosis of FBPase deficiency, using cultured monocytes as a source for FBPase mRNA. In the present study, we confirmed the presence of the same genetic mutation in this patient by amplifying genomic DNA. Molecular analysis was also performed to diagnose another 12 Japanese patients with FBPase deficiency. Four mutations responsible for FBPase deficiency were identified in 10 patients from 8 unrelated families among a total of 13 patients from 11 unrelated families; no mutation was found in the remaining 3 patients from 3 unrelated families. The identified mutations included the mutation reported earlier, with an insertion of one G residue at base 961 in exon 7 (960/961insG) (10 alleles, including 2 alleles in the Japanese family from our previous report [46% of the 22 mutant alleles]), and three novel mutations--a G-->A transition at base 490 in exon 4 (G164S) (3 alleles [14%]), a C-->A transversion at base 530 in exon 4 (A177D) (1 allele [4%]), and a G-->T transversion at base 88 in exon 1 (E30X) (2 alleles [9%]). FBPase proteins with G164S or A177D mutations were enzymatically inactive when purified from E. coli. Another new mutation, a T-->C transition at base 974 in exon 7 (V325A), was found in the same allele with the G164S mutation in one family (one allele) but was not responsible for FBPase deficiency. Our results indicate that the insertion of one G residue at base 961 was associated with a preferential disease-causing alternation in 13 Japanese patients. Our results also indicate accurate carrier detection in eight families (73%) of 11 Japanese patients with FBPase deficiency, in whom mutations in both alleles were identified.     

Canavan disease: mutations among Jewish and non-jewish patients.

Canavan disease is an autosomal recessive leukodystrophy caused by the deficiency of aspartoacylase (ASPA). Sixty-four probands were analyzed for mutations in the ASPA gene. Three point mutations--693C-->A, 854A-->C, and 914C-->A--were identified in the coding sequence. The 693C-->A and 914C-->A base changes, resulting in nonsense tyr231-->ter and missense ala305-->glu mutations, respectively, lead to complete loss of ASPA activity in in vitro expression studies. The 854A-->C transversion converted glu to ala in codon 285. The glu285-->ala mutant ASPA has 2.5% of the activity expressed by the wild-type enzyme. A fourth mutation, 433 --2(A-->G) transition, was identified at the splice-acceptor site in intron 2. The splice-site mutation would lead to skipping of exon 3, accompanied by a frameshift, and thus would produce aberrant ASPA. Of the 128 unrelated Canavan chromosomes analyzed, 88 were from probands of Ashkenazi Jewish descent. The glu285-->ala mutation was predominant (82.9%) in this population, followed by the tyr231-->ter (14.8%) and 433 --2(A-->G) (1.1%) mutations. The three mutations account for 98.8% of the Canavan chromosomes of Ashkenazi Jewish origin. The ala305-->glu mutation was found exclusively in non-Jewish probands of European descent and constituted 60% of the 40 mutant chromosomes. Predominant occurrence of certain mutations among Ashkenazi Jewish and non-Jewish patients with Canavan disease would suggest a founding-father effect in propagation of these mutant chromosomes.     

Complete deletion of the proteolipid protein gene (PLP) in a family with X-linked Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is an X-linked neurologic disorder characterized by dysmyelination in the central nervous system. Proteolipid protein (PLP), a major structural protein of myelin, is coded on the X chromosome. It has been postulated that a defect in the PLP gene is responsible for PMD. Different single-nucleotide substitutions have been found in conserved regions of the PLP gene of four unrelated PMD patients. Novel Southern blot patterns suggested a complex rearrangement in a fifth family. Linkage to PLP has been shown in others. We evaluated the PLP locus in a four-generation family with two living males affected with X-linked PMD. Analysis of DNA from the affected males revealed complete absence of a band, with PLP probes encompassing the promoter region, the entire coding region, and the 3' untranslated region and spanning at least 29 kb of genomic DNA. DNA from unaffected relatives gave the expected band pattern. Two obligate and one probable carrier women were hemizygous for the PLP locus by dosage analysis. Although it is unlikely, the previously described point mutations in PLP could represent polymorphisms. The finding of complete deletion of the PLP gene in our family is a stronger argument that mutations in PLP are responsible for X-linked PMD.     

Identification and Functional Analysis of GJA8 Mutation in a Chinese Family with Autosomal Dominant Perinuclear Cataracts

Congenital cataract is a clinically and genetically heterogeneous group of eye disorders that causes visual impairment and childhood blindness. The purpose of this study was to identify the genetic defect associated with autosomal dominant congenital perinuclear cataract in a Chinese family. A detailed family history and clinical data of the family were recorded, and candidate gene sequencing was performed to screen for mutation-causing disease in our study. Direct sequencing revealed a c.601G>A (p.E201K) transversion in exon 2 of GJA8. This mutation co-segregated with all affected individuals in the family and was not found in unaffected family members or 100 unrelated controls. The function and mechanism of novel GJA8 point mutation E201K in Chinese patients were then investigated in this study. We found E201K aberrantly located in cytoplasm and prevented its location in the plasma membrane. Induction of E201K expression led to a decrease in cell growth and viability by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Our study provides important evidence that GJA8 is a disease-causing gene for congenital cataract and that mutation of GJA8 has a potential causative effect.     

Identification of missense, nonsense, and deletion mutations in the GRHPR gene in patients with primary hyperoxaluria type II (PH2)

Primary hyperoxaluria type II (PH2) is a rare disease characterized by the absence of an enzyme with glyoxylate reductase, hydroxypyruvate reductase, and D-glycerate dehydrogenase activities. The gene encoding this enzyme (GRHPR) has been characterized, and a single mutation has been detected in four PH2 patients. In this report, we have identified five novel mutations. One nonsense mutation (C295T) results in a premature stop codon at codon 99. A 4-bp deletion mutation has been found in the 5' consensus splice site of intron D, resulting in a predicted splicing error. Three missense mutations have been detected, including a missense transversion (T965G) in exon 9 (Met322Arg), a missense transition (G494A) in the putative co-factor binding site in exon 6 (Gly165Asp), and a substitution of an adenosine for a guanine in the 3' splice site of intron G. The functional consequences of the missense transversion and transition mutations have been investigated by transfection of cDNA encoding the mutated protein into COS cells. Cells transfected with either mutant construct have no enzymatic activity, a finding that is not significantly different from the control (empty) vector (P<0.05). These results further confirm that mutations in the GRHPR gene form the genetic basis of PH2. Ten of the 11 patients that we have genotyped are homozygous for one of the six mutations identified to date. Because of this high proportion of homozygotes, we have used microsatellite markers in close linkage with the GRHPR gene to investigate the possibility that the patients are the offspring of related individuals. Our data suggest that two thirds of our patients are the offspring of either closely or distantly related persons. Furthermore, genotyping has revealed the possible presence of a founder effect for the two most common mutations and the location of the gene near the marker D9S1874.     

Pelizaeus-Merzbacher disease: identification of Xq22 proteolipid-protein duplications and characterization of breakpoints by interphase FISH.

Pelizaeus-Merzbacher disease (PMD) is an X-linked, dysmyelinating disorder of the CNS. Duplications of the proteolipid protein (PLP) gene have been found in a proportion of patients, suggesting that, in addition to coding-region or splice-site mutations, overdosage of the gene can cause PMD. We show that the duplication can be detected by interphase FISH, using a PLP probe in five patients and their four asymptomatic carrier mothers. The extent of the duplication was analyzed in each family by interphase FISH, with probes from a 1. 7-Mb region surrounding the PLP gene between markers DXS83 and DXS94. A large duplication >=500 kb was detected, with breakpoints that differed, between families, at the proximal end. Distinct separation of the duplicated PLP signals could be seen only on metaphase chromosomes in one family, providing further evidence that different duplication events are involved. Quantitative fluorescent multiplex PCR was used to confirm the duplication in patients, by the detection of increased copy number of the PLP gene. Multiallelic markers from the duplicated region were analyzed, since the identification of two alleles in an affected boy would indicate a duplication. The majority of boys were homozygous for all four markers, compared with their mothers, who were heterozygous for one to three of the markers. These results suggest that intrachromosomal rearrangements may be a common mechanism by which duplications arise in PMD. One boy was heterozygous for the PLP marker, indicating a duplication and suggesting that interchromosomal rearrangements of maternal origin also can be involved. Since duplications are a major cause of PMD, we propose that interphase FISH is a reliable method for diagnosis and identification of female carriers.     

Novel three missense mutations observed in Von Hippel-Lindau gene in a patient reported with renal cell carcinoma

Von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary cancer syndrome that predisposes to the development of a variety of benign and malignant tumors, especially cerebellar hemangioblastomas, retinal angiomas and clear-cell renal cell carcinomas (RCC). We have identified of VHL gene using immunohistochemistry in a patient who was diagnosed for RCC. In order to understand the involvement of mutation in the VHL gene exon 1 was amplified and sequenced (accession number: JX 401534). The sequence analysis revealed the presence of novel missense mutations c.194 C>T, c.239 G>A, c.278 G>A, c.319 C>G, c. 337 C > G leading to the following variations p.Ala 65 Val, p.Gly 80 Asp, p.Gly 93 Glu, p.Gln 107 Glu, p.Gln 113 Glu in the protein.     

Exome Sequencing and Linkage Analysis Identified Tenascin-C (TNC) as a Novel Causative Gene in Nonsyndromic Hearing Loss

In this study, a five-generation Chinese family (family F013) with progressive autosomal dominant hearing loss was mapped to a critical region spanning 28.54 Mb on chromosome 9q31.3-q34.3 by linkage analysis, which was a novel DFNA locus, assigned as DFNA56. In this interval, there were 398 annotated genes. Then, whole exome sequencing was applied in three patients and one normal individual from this family. Six single nucleotide variants and two indels were found co-segregated with the phenotypes. Then using mass spectrum (Sequenom, Inc.) to rank the eight sites, we found only the TNC gene be co-segregated with hearing loss in 53 subjects of F013. And this missense mutation (c.5317G>A, p.V1773M ) of TNC located exactly in the critical linked interval. Further screening to the coding region of this gene in 587 subjects with nonsyndromic hearing loss (NSHL) found a second missense mutation, c.5368A>T (p. T1796S), co-segregating with phenotype in the other family. These two mutations located in the conserved region of TNC and were absent in the 387 normal hearing individuals of matched geographical ancestry. Functional effects of the two mutations were predicted using SIFT and both mutations were deleterious. All these results supported that TNC may be the causal gene for the hearing loss inherited in these families. TNC encodes tenascin-C, a member of the extracellular matrix (ECM), is present in the basilar membrane (BM), and the osseous spiral lamina of the cochlea. It plays an important role in cochlear development. The up-regulated expression of TNC gene in tissue repair and neural regeneration was seen in human and zebrafish, and in sensory receptor recovery in the vestibular organ after ototoxic injury in birds. Then the absence of normal tenascin-C was supposed to cause irreversible injuries in cochlea and caused hearing loss.     

Paralytic Tremor (pt): A New Allele of the Proteolipid Protein Gene in Rabbits

Abstract: Paralytic tremor ( pt ) is a sex-linked mutation in rabbit that affects myelination of the CNS. Myelin in the pt brains represents ∼30% of the normal levels. Previously we showed that the pt mutation affects primarily proteolipid protein ( Plp ) gene expression. In the present study we investigated the relative effect of the pt mutation on two distinctive Plp gene products, PLP- and DM-20-specific messenger RNAs. Our results showed that both PLP and DM-20 are affected and that the ratio DM-20/PLP was higher in pt rabbits than in age-matched controls. We sequenced normal rabbit PLP cDNA and characterized pt mutation at the DNA level. Rabbit PLP sequence, deduced from cDNA, differs from the human protein only at Thr¹⁹⁸. Sequence analysis of the mutant cDNA revealed a transversion T → A in exon 2 of the Plp gene. This point mutation, which is placed at the end of the first potential transmembrane domain, results in a substitution of His³⁶ by a glutamine. This transversion abolishes a restriction site that enabled us to screen a large number of animals and observe a perfect correlation between the pt allele and the abnormal phenotype.     

Molecular diagnostics for myelin proteolipid protein gene mutations in Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is a clinically heterogeneous, slowly progressive leukodystrophy. The recent detection of mutations in the myelin proteolipid protein (PLP) gene in several PMD patients offers the opportunity both to design DNA-based tests that would be useful in diagnosing a proportion of PMD cases and, in particular, to evaluate the diagnostic utility of single-strand conformation polymorphism (SSCP) analysis for this disease. A combination of SSCP analysis and direct sequencing of PCR-amplified DNA was used to screen for PLP mutations in 24 patients affected with leukodystrophies of unknown etiology. Two heretofore undescribed mutations in the PLP gene were identified, Asp202His in exon 4 and Gly73Arg in exon 3. The ease and efficiency of SSCP analysis in detecting new mutations support the utilization of this technique in screening for PLP mutations in patients with unexplained leukodystrophies.     

Whole Exome Sequencing Reveals Homozygous Mutations in RAI1, OTOF,and SLC26A4 Genes Associated with Nonsyndromic Hearing Loss in Altaian Families (South Siberia)

Hearing loss (HL) is one of the most common sensorineural disorders and several dozen genes contribute to its pathogenesis. Establishing a genetic diagnosis of HL is of great importance for clinical evaluation of deaf patients and for estimating recurrence risks for their families. Efforts to identify genes responsible for HL have been challenged by high genetic heterogeneity and different ethnic-specific prevalence of inherited deafness. Here we present the utility of whole exome sequencing (WES) for identifying candidate causal variants for previously unexplained nonsyndromic HL of seven patients from four unrelated Altaian families (the Altai Republic, South Siberia). The WES analysis revealed homozygous missense mutations in three genes associated with HL. Mutation c.2168A>G (SLC26A4) was found in one family, a novel mutation c.1111G>C (OTOF) was revealed in another family, and mutation c.5254G>A (RAI1) was found in two families. Sanger sequencing was applied for screening of identified variants in an ethnically diverse cohort of other patients with HL (n = 116) and in Altaian controls (n = 120). Identified variants were found only in patients of Altaian ethnicity (n = 93). Several lines of evidences support the association of homozygosity for discovered variants c.5254G>A (RAI1), c.1111C>G (OTOF), and c.2168A>G (SLC26A4) with HL in Altaian patients. Local prevalence of identified variants implies possible founder effect in significant number of HL cases in indigenous population of the Altai region. Notably, this is the first reported instance of patients with RAI1 missense mutation whose HL is not accompanied by specific traits typical for Smith-Magenis syndrome. Presumed association of RAI1 gene variant c.5254G>A with isolated HL needs to be proved by further experimental studies.     

A point mutation at the X-chromosomal proteolipid protein locus in Pelizaeus-Merzbacher disease leads to disruption of myelinogenesis.

A group of inherited neurological disorders are the X-chromosome linked dysmyelinoses, in which myelin membranes of the CNS are missing or perturbed due to a strongly reduced number of differentiated oligodendrocytes. In animal dysmyelinoses (jimpy mouse, msd-mouse, md rat, shaking pup) mutations of the main integral myelin membrane protein, proteolipid protein, have been identified. Pelizaeus-Merzbacher disease (PMD) or sudanophilic leucodystrophy is an X-linked dysmyelinosis in humans. We report here on the molecular basis of the defect of affected males of a PMD kindred. Rearrangements of the PLP gene were excluded by Southern blot hybridisation analysis and PCR amplification of overlapping domains of the PLP gene. Sequence analysis revealed one single C----T transition in exon IV, which leads to a threonine----isoleucine substitution within a hydrophobic intramembrane domain. The impact of this amino-acid exchange on the structure of PLP in the affected cis membrane domain is discussed. A space filling model of this domain suggests a tight packing of the alpha-helices of the loop which is perturbed by the amino-acid substitution in this PMD exon IV mutant. The C----T transition in exon IV abolishes a Hph I restriction site. This mutation at the recognition site for Hph I (RFLP) and allele-specific primers have been used for mutation screening the PMD kindred.