Identification of the gene FMR2, associated with FRAXE mental retardation.

Five folate-sensitive fragile sites have been characterized at the molecular level (FRAXA, FRAXE, FRAXF, FRA16A and FRA11B). Three of them (FRAXA, FRAXE and FRA11B) are associated with clinical problems, and two of the genes (FMR1 in FRAXA and CBL2 in FRA11B) have been identified. All of these fragile sites are associated with (CCG)n/(CGG)n triplet expansions which are hypermethylated beyond a critical size. FRAXE is a rare folate sensitive fragile site only recently recognized. Its cytogenetic expression was found to involve the amplification of a (CCG)n repeat adjacent to a CpG island. Normal alleles vary from 6 to 25 copies. Expansions of greater than 200 copies were found in FRAXE expressing males and their FRAXE associated CpG island was fully methylated. An association of FRAXE expression with concurrent methylation of the CpG island and mild non-specific mental handicap in males has been reported by several groups. We now report the cloning and characterization of a gene (FMR2) adjacent to FRAXE. Elements of FMR2 were initially identified from sequences deleted from a developmentally delayed boy. We correlate loss of FMR2 expression with (CCG)n expansion at FRAXE, demonstrating that this is a gene associated with the CpG island adjacent to FRAXE and contributes for FRAXE-associated mild mental retardation.

A novel X-linked gene, G4.5. is responsible for Barth syndrome.

Barth syndrome is a severe inherited disorder, often fatal in childhood, characterized by cardiac and skeletal myopathy, short stature and neutropenia. The disease has been mapped to a very gene-rich region in distal portion of Xq28. We now report the identification of unique mutations in one of the genes in this region, termed G4.5, expressed at high level in cardiac and skeletal muscle. Different mRNAs can be produced by alternative splicing of the primary G4.5 transcript, encoding novel proteins that differ at the N terminus and in the central region. The mutations introduce stop codons in the open reading frame interrupting translation of most of the putative proteins (which we term 'tafazzins'). Our results suggest that G4.5 is the genetic locus responsible for the Barth syndrome.

Identification of the gene (SEDL) causing X-linked spondyloepiphyseal dysplasia tarda.

Spondyloepiphyseal dysplasia tarda (SEDL; MIM 313400) is an X-linked recessive osteochondrodysplasia that occurs in approximately two of every one million people. This progressive skeletal disorder which manifests in childhood is characterized by disproportionate short stature with short neck and trunk, barrel chest and absence of systemic complications. Distinctive radiological signs are platyspondyly with hump-shaped central and posterior portions, narrow disc spaces, and mild to moderate epiphyseal dysplasia. The latter usually leads to premature secondary osteoarthritis often requiring hip arthroplasty. Obligate female carriers are generally clinically and radiographically indistinguishable from the general population, although some cases have phenotypic changes consistent with expression of the gene defect. The SEDL gene has been localized to Xp22 (refs 8,9) in the approximately 2-Mb interval between DXS16 and DXS987 (ref. 10). Here we confirm and refine this localization to an interval of less than 170 kb by critical recombination events at DXS16 and AFMa124wc1 in two families. In one candidate gene we detected three dinucleotide deletions in three Australian families which effect frameshifts causing premature stop codons. The gene designated SEDL is transcribed as a 2.8-kb transcript in many tissues including fetal cartilage. SEDL encodes a 140 amino acid protein with a putative role in endoplasmic reticulum (ER)-to-Golgi vesicular transport.

Fragile X syndrome without CCG amplification has an FMR1 deletion.

We describe a patient with typical clinical features of the fragile X syndrome, but without cytogenetic expression of the fragile X or an amplified CCG trinucleotide repeat fragment. The patient has a previously uncharacterized submicroscopic deletion encompassing the CCG repeat, the entire FMR1 gene and about 2.5 megabases of flanking sequences. This finding confirms that the fragile X phenotype can exist, without amplification of the CCG repeat or cytogenetic expression of the fragile X, and that fragile X syndrome is a genetically homogeneous disorder involving FMR1. We also found random X-inactivation in the mother of the patient who was shown to be a carrier of this deletion.

Mutations in GDI1 are responsible for X-linked non-specific mental retardation.

Rab GDP-dissociation inhibitors (GDI) are evolutionarily conserved proteins that play an essential role in the recycling of Rab GTPases required for vesicular transport through the secretory pathway. We have found mutations in the GDI1 gene (which encodes uGDI) in two families affected with X-linked non-specific mental retardation. One of the mutations caused a non-conservative substitution (L92P) which reduced binding and recycling of RAB3A, the second was a null mutation. Our results show that both functional and developmental alterations in the neuron may account for the severe impairment of learning abilities as a consequence of mutations in GDI1, emphasizing its critical role in development of human intellectual and learning abilities.

Characterization of new PCR based markers for mapping and diagnosis: AC dinucleotide repeat markers at the DXS237 (GMGX9) and DXS102 (cX38.1) loci.

Genomic insert DNAs from 45 probes representing 113.4 kb of the X chromosome were screened for AC dinucleotide repeat sequence. Two new AC repeat sequences were identified with length polymorphism based on variation in repeat copy number. One at DXS237 exhibits 44% heterozygosity and is potentially useful for rapid diagnosis and mapping of X-linked disorders in Xp22.3. The other, at DXS102 in Xq26, has 71% heterozygosity. This marker will improve accuracy of diagnoses by linkage for families with Börjeson-Forssman-Lehmann syndrome. Review of the literature has identified 31 PCR based markers on the X chromosome, with minimum heterozygosity of 50%, applicable to the mapping and diagnosis of X-linked disorders.

Use of linkage data obtained in single families: prenatal diagnosis of a new X-linked mental retardation syndrome.

Prenatal diagnosis was requested by an obligate carrier of a new syndrome of X-linked mental retardation. There was close linkage between the disease gene and the hypervariable VNTR marker DXS255 with a lod score of 4.82 at o = 0 (90% support interval 0.00-0.12). When the request for prenatal diagnosis was made, additional family members were examined, resulting in an amended lod score of 6.71 at o = 0.0 (90% support interval 0.00-0.09). There were no informative flanking markers at the time of the request for prenatal diagnosis; hence it proceeded by 2 point linkage analysis. The fetus was female with a carrier risk in the interval of 91-100%. Given the limitations of the mapping data available for this disorder at the time of the request, the options of accepting or rejecting this as a case for prenatal diagnosis were carefully considered. Whilst prenatal diagnosis based on fetal sexing would be sufficient to prevent the birth of an affected child, the magnitude of the known two-point lod score between DXS255 and the disease gene provided a means for diagnosis with an accuracy between 91 and 100%.

Localisation of the gene for X-linked reticulate pigmentary disorder with systemic manifestations (PDR), previously known as X-linked cutaneous amyloidosis.

X-linked reticulate pigmentary disorder (PDR), previously reported as X-linked cutaneous amyloidosis (MIM#301220), is characterized by brown pigmentation of the skin which follows the lines of Blaschko in females but appears as reticulate sheets in males. Males may suffer severe gastrointestinal disorders in infancy with failure to thrive and early death. Nowadays symptomatic treatment allows survival and other manifestations may appear such as corneal dystrophy with severe photophobia or chronic respiratory disease. Amyloid deposition in the skin may be no more than an age-dependent secondary manifestation. The PDR gene was localised by linkage analysis to Xp21-p22. The background genetic map is Xpter-DXS996-22.5-DXS207-3.3-DXS999-3.3-DXS36 5-14.2-DXS989-4.1-3'DMD-3.5- DXS997-1.0-STR44-9.3-DYSI-2.3-DXS1068-11.0-DX S228 with distances between markers given in cM. Recombinants detected with DXS999 distally and DXS228 proximally, define the limits to the localisation. Linkage was found with several markers within this interval. Peak lod scores of 3.21 at theta = 0.0 were obtained between PDR and DXS989 and between PDR and 5'DYSI within the dystrophin locus.

Genetic localisation of MRX27 to Xq24-26 defines another discrete gene for non-specific X-linked mental retardation.

A large family with non-specific X-linked mental retardation (MRX) was first described in 1991 [Glass et al., 1991], with a suggestion of linkage to Xq26-27. The maximum lod score was 1.60 (theta = 0.10) with the F9 locus. The localisation of this MRX gene has now been established by linkage to microsatellite markers. Peak pairwise lod scores of 4.02 and 4.01 (theta = 0.00) were attained at the DXS1114 and DXS994 loci respectively. This MRX gene is now designated MRX27 and is localised to Xq24-26 by recombination events detected by DXS424 and DXS102. This regional localisation spans 26.2 cM on the genetic background map and defines another distinct MRX interval by linkage to a specific region of the X chromosome.

Linkage and genetic counselling for the fragile X using DNA probes 52A, F9, DX13, and ST14.

Linkage data using the markers DXS51, F9, DXS15, and DXS52 are presented from 14 pedigrees segregating with the fragile X. Cytogenetic and DNA data were combined by two- or three-point linkage analysis for estimation of lod scores and carrier probabilities in potential carriers. Recombination frequencies (theta) corresponding to maximum z scores (zeta) were obtained for DXS51 (zeta = 3.45, theta = 0.0), DXS15 (zeta = 0.40, theta = 0.06), F9 (zeta = 3.15, theta = 0.09), and DXS52 (zeta = 3.60, theta = 0.11) with the fragile X. Considerable alterations to carrier probabilities occurred in some cases, especially when flanking markers were informative. The chance of mentally impaired offspring was reduced to 1% for five of eight women with prior carrier probabilities of 32%. Three pedigrees were identified in which mutation had possibly occurred. An alternative explanation for two of these was inheritance of the fragile X from normal males and for the other inheritance from a clinically normal woman. Probabilities were computed for each of these alternatives.

Linkage studies with the gene for an X-linked syndrome of mental retardation, microcephaly and spastic diplegia (MRX2)

A family in which a gene (MRX2) is segregating for an X-linked syndrome of mental retardation, short stature, microcephaly, brachycephaly, spastic diplegia, small testes and possible intra-uterine growth retardation is described. There are 7 clearly affected males and one possibly affected infant in the family. The obligate carriers are normal. Linkage studies show a suggestion of linkage to loci near the centromere. The maximum lod score was 2.10 at theta = 0.11 for DXYS1, assuming the possibly affected male carried the MRX2 gene. There were lower lod scores suggestive of linkage with DXS7 (theta = 0.14; z = 1.29) and DXS94 (theta = 0.11; z = 1.22).

Barth syndrome: clinical features and confirmation of gene localisation to distal Xq28.

Barth syndrome is an X-linked disorder characterised by cardioskeletal myopathy of variable severity usually fatal in childhood, and neutropenia. We ascertained a large pedigree with affected males in 3 generations. All affected males had dilated cardiomyopathy, with endocardial fibroelastosis (EFE) in some. The locus for Barth syndrome in this family was found to be closely linked to DXS52 (z = 2.78, theta = 0.0). The family was nonrecombinant for DXS52 in distal Xq28, but recombinant for DXS374 which maps proximal to DXS52. This localised Barth syndrome distal to DXS374, confirming a previous localisation to distal Xq28. As yet there is no evidence for genetic heterogeneity of Barth syndrome.

Regional localisation of a non-specific X-linked mental retardation gene (MRX19) to Xp22.

A gene responsible for a non-specific form of X-linked mental retardation (MRX19) was localised by linkage analysis. Exclusions and regional localisation were made using 21 highly informative PCR-based markers along the X chromosome. Significant lod scores at a recombination fraction of zero were detected with the marker loci DXS207, DXS987 (Zmax = 3.58) and DXS999 (Zmax = 3.28) indicating that this gene is localised to the proximal portion of Xp22. Recombination between MRX19 and the flanking loci KAL and DXS989 was observed. The multipoint CEPH background map, with map distances in cM, is DXS996-1.8-KAL-19.0-DXS207-0.9-[DXS987,DXS443 ]-4.3-DXS999-3.5-DXS365-14.0-DXS989. Two other MRX disorders and two syndromal mental retardations, Coffin-Lowry syndrome and Partington syndrome, have been mapped to this region. There is a possibility that the 3 MRX disorders are the same entity. Most MRX disorders remain clustered around the pericentromeric region.

Refinement of the background genetic map of Xq26-q27 and gene localisation for Börjeson-Forssman-Lehmann Syndrome.

A detailed map of genetic markers was constructed around the gene for the X-linked mental retardation syndrome of Börjeson-Forssman-Lehmann (BFLS). A multipoint linkage map of framework markers across Xq26-27, based on CEPH families, was integrated with the physical map, based on a YAC contig, to confirm marker order. The remaining genetic markers, which could not be ordered by linkage, were added to create the comprehensive genetic back-ground map, in the order determined by physical mapping, to determine genetic distances between adjacent markers. This background genetic map is applicable to the refinement of the regional localisation for any disease gene mapping to this region. The BFLS gene was localised using this background map in an extended version of the family described by Turner et al. [1989]. The regional localisation for BFLS extends between recombination events at DXS425 and DXS105, an interval of 24.6 cM on the background genetic map. The phenotypic findings commonly seen in the feet of affected males and obligate carrier females may represent a useful clinical indicator of carrier status in potential female carriers in the family. Recombination between DXS425 and DXS105 in a female with such characteristic feet suggests that the distal limit of the regional localisation for the BFLS gene might reasonably be reduced to DXS294 for the purpose of selecting candidate genes, reducing the interval for the BFLS gene to 15.5 cM. Positional candidate genes from the interval between DXS425 and DXS105 include the SOX3 gene, mapped between DXS51(52A) and DXS98(4D-8). SOX3 may have a role in regulating the development of the nervous system. The HMG-box region of this single exon gene was examined by PCR for a deletion and then sequenced. No deviation from normal was observed, excluding mutations in the conserved HMG-box region as the cause of BFLS in this family.

Predictive diagnosis for polycystic kidney disease using DNA markers.

Two families in which the gene for the common, autosomal dominant form of polycystic kidney disease (PKD1) was present were examined using flanking DNA markers. The 5'HVR probe detects a linked DNA marker 8 map units distal to the PKD1 gene in males and 1 unit distal to the PKD1 gene in females. The 24-1 probe detects another linked DNA marker 4 map units proximal to the PKD1 gene in males and 0.5 map units proximal to the PKD1 gene in females. When each marker is informative they can be used as a pair flanking the disease gene to follow accurately its transmission through families for presymptomatic or prenatal prediction. For an asymptomatic individual tested in one family, DNA studies reduced the 50% prior risk of carrying the disease gene to 0.006%. An affected woman in a second family was shown to be fully informative for the flanking markers. In a future pregnancy, it will be possible to modify the 50% prior fetal risk to either 0.008% or 99.99% depending on which maternal chromosome 16 is transmitted, and provided that no cross-over occurs between the flanking markers (probability, 1.5%).

Genotype-phenotype relationships in fragile X syndrome: a family study.

Relationships between the measures of intellectual and physical status in the fragile X syndrome and the size of amplification of the fragile X-specific fragment, equivalent to the number of CCG repeats within the FMR1 locus, were studied by a maximum-likelihood scoring technique for analysis of pedigree data. This allows for estimation of random effects (genetic and environmental variance) concurrently with other (fixed) effects in a quantitative trait. FMR1 expression is usually shut down in males penetrant for the fragile X syndrome who have hypermethylated CCG amplifications of > or = 0.6 kb. The assumption of the step versus curvilinear function representing this relationship was tested by the likelihood-ratio criterion. The maximum-likelihood parameters were based on the most appropriate model for each measure. The results were indicative of the presence of a curvilinear relationship between the amplification size and the two intellectual scores, the Peabody Picture Vocabulary Test and Block Design Test, measuring verbal and spatial abilities, respectively. Reasons for the unexpected curvilinear regression between the amplification size and intellectual scores were explained further by methylation analysis of fragile X males with amplifications of 0.6 < delta < or = 1.2 kb who appeared to be responsible for the curvilinearity of the relationship. Four of these showed unmethylated status of the amplified bands in lymphocytes, which were presumably transcriptionally active. Removal of the aberrant individuals led to the anticipated step function between amplification and intellectual scores. For the combined anthropometric score, as well as for several single physical measures, the step function was the most appropriate model regardless of the inclusion or omission of the aberrant individuals in the pedigree sample.

Direct molecular diagnosis of myotonic dystrophy.

Myotonic dystrophy (DM) arises from an unstable trinucleotide (CTGn) repeat sequence within the DM locus at 19q13.3. Twenty-three myotonic dystrophy families containing 205 persons with no symptoms, minimal manifestations, classic DM or congenital DM were investigated to validate the application of the pM10M6 probe to direct molecular diagnosis. Affected family members had been diagnosed clinically and the unaffected family members had been assigned carrier probabilities close to either zero or 100%, using closely linked flanking markers. Southern analysis identified all 89 DM gene carriers as having expansions of the unstable element. PstI detected all small expansions of the repeat sequence as easily seen discrete bands; but large expansions were usually seen as diffuse smears, sometimes difficult to distinguish from lane background. EcoRI concentrated these diffuse smears, associated with somatic instability, into discrete bands which were easy to detect; but it did not resolve the smaller expansions present in 9 (10%) of the DM carriers. It is essential that PstI and EcoRI gels are run in parallel to detect all DM gene carriers. The extent of expansion of CTG correlated with age of onset and disease severity. Biopsies of various fetal tissues from two terminated pregnancies confirmed the diagnosis obtained by CVS and revealed no heterogeneity between tissues at this developmental stage. Further expansion occurred during the culture of CVS cells, indicating that direct prenatal diagnosis needs to be carried out on CVS tissue rather than on cultured cells. The intergenerational change of the repeat sequence from DM parent to DM offspring showed a significant parental sex difference for those parents with large expansions. Contraction of the unstable element was observed in the three males carrying the largest expansions and could explain why congenital DM is exclusively of maternal origin.

Predictive diagnosis of myotonic dystrophy with flanking microsatellite markers.

Linkage was shown between the myotonic dystrophy locus (DM) and a highly polymorphic AC repeat marker within the kallikrein (KLK1) locus (Z = 3.00, theta = 0.0). Linkage between KLK1 and the highly polymorphic AC repeat marker within the apolipoprotein C2 (APOC2) locus, which had been established in normal families, was confirmed in myotonic dystrophy families (Z = 4.37, theta = 0.11). These highly polymorphic AC repeat markers flank DM on chromosome 19. The gene order is cen-APOC2 (0.03) DM (0.08) KLK1-qter with recombination frequencies shown in parentheses. Genotypes for the AC repeat markers can be determined simultaneously by multiplex PCR and separation of the two base pair differences between adjacent alleles on sequencing gels. In informative families, this approach provides rapid diagnosis and is more accurate than methods using markers restricted to the proximal side of the myotonic dystrophy gene.