Karen Woodward

Over- and Underdosage of SOX3 Is Associated with Infundibular Hypoplasia and Hypopituitarism

Duplications of Xq26-27 have been implicated in the etiology of X-linked hypopituitarism associated with mental retardation (MR). Additionally, an expansion of a polyalanine tract (by 11 alanines) within the transcription factor SOX3 (Xq27.1) has been reported in patients with growth hormone deficiency and variable learning difficulties. We report a submicroscopic duplication of Xq27.1, the smallest reported to date (685.6 kb), in two siblings with variable hypopituitarism, callosal abnormalities, anterior pituitary hypoplasia (APH), an ectopic posterior pituitary (EPP), and an absent infundibulum. This duplication contains SOX3 and sequences corresponding to two transcripts of unknown function; only Sox3 is expressed in the infundibulum in mice. Next, we identified a novel seven-alanine expansion within a polyalanine tract in SOX3 in a family with panhypopituitarism in three male siblings with an absent infundibulum, severe APH, and EPP. This mutation led to reduced transcriptional activity, with impaired nuclear localization of the mutant protein. We also identified a novel polymorphism (A43T) in SOX3 in another child with hypopituitarism. In contrast to findings in previous studies, there was no evidence of MR or learning difficulties in our patients. We conclude that both over- and underdosage of SOX3 are associated with similar phenotypes, consisting of infundibular hypoplasia and hypopituitarism but not necessarily MR.

Exon array CGH: detection of copy-number changes at the resolution of individual exons in the human genome.

The development of high-throughput screening methods such as array-based comparative genome hybridization (array CGH) allows screening of the human genome for copy-number changes. Current array CGH strategies have limits of resolution that make detection of small (less than a few tens of kilobases) gains or losses of genomic DNA difficult to identify. We report here a significant improvement in the resolution of array CGH, with the development of an array platform that utilizes single-stranded DNA array elements to accurately measure copy-number changes of individual exons in the human genome. Using this technology, we screened 31 patient samples across an array containing a total of 162 exons for five disease genes and detected copy-number changes, ranging from whole-gene deletions and duplications to single-exon deletions and duplications, in 100% of the cases. Our data demonstrate that it is possible to screen the human genome for copy-number changes with array CGH at a resolution that is 2 orders of magnitude higher than that previously reported.

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.

Heterogeneous Duplications in Patients with Pelizaeus-Merzbacher Disease Suggest a Mechanism of Coupled Homologous and Nonhomologous Recombination

We describe genomic structures of 59 X-chromosome segmental duplications that include the proteolipid protein 1 gene (PLP1) in patients with Pelizaeus-Merzbacher disease. We provide the first report of 13 junction sequences, which gives insight into underlying mechanisms. Although proximal breakpoints were highly variable, distal breakpoints tended to cluster around low-copy repeats (LCRs) (50% of distal breakpoints), and each duplication event appeared to be unique (100 kb to 4.6 Mb in size). Sequence analysis of the junctions revealed no large homologous regions between proximal and distal breakpoints. Most junctions had microhomology of 1-6 bases, and one had a 2-base insertion. Boundaries between single-copy and duplicated DNA were identical to the reference genomic sequence in all patients investigated. Taken together, these data suggest that the tandem duplications are formed by a coupled homologous and nonhomologous recombination mechanism. We suggest repair of a double-stranded break (DSB) by one-sided homologous strand invasion of a sister chromatid, followed by DNA synthesis and nonhomologous end joining with the other end of the break. This is in contrast to other genomic disorders that have recurrent rearrangements formed by nonallelic homologous recombination between LCRs. Interspersed repetitive elements (Alu elements, long interspersed nuclear elements, and long terminal repeats) were found at 18 of the 26 breakpoint sequences studied. No specific motif that may predispose to DSBs was revealed, but single or alternating tracts of purines and pyrimidines that may cause secondary structures were common. Analysis of the 2-Mb region susceptible to duplications identified proximal-specific repeats and distal LCRs in addition to the previously reported ones, suggesting that the unique genomic architecture may have a role in nonrecurrent rearrangements by promoting instability.

Additional Copies of the Proteolipid Protein Gene Causing Pelizaeus-Merzbacher Disease Arise by Separate Integration into the X Chromosome

The proteolipid protein gene (PLP) is normally present at chromosome Xq22. Mutations and duplications of this gene are associated with Pelizaeus-Merzbacher disease (PMD). Here we describe two new families in which males affected with PMD were found to have a copy of PLP on the short arm of the X chromosome, in addition to a normal copy on Xq22. In the first family, the extra copy was first detected by the presence of heterozygosity of the AhaII dimorphism within the PLP gene. The results of FISH analysis showed an additional copy of PLP in Xp22.1, although no chromosomal rearrangements could be detected by standard karyotype analysis. Another three affected males from the family had similar findings. In a second unrelated family with signs of PMD, cytogenetic analysis showed a pericentric inversion of the X chromosome. In the inv(X) carried by several affected family members, FISH showed PLP signals at Xp11.4 and Xq22. A third family has previously been reported, in which affected members had an extra copy of the PLP gene detected at Xq26 in a chromosome with an otherwise normal banding pattern. The identification of three separate families in which PLP is duplicated at a noncontiguous site suggests that such duplications could be a relatively common but previously undetected cause of genetic disorders.

Proteolipid protein gene: Pelizaeus-Merzbacher disease in humans and neurodegeneration in mice.

The dosage of the myelin gene and mutant forms of the protein can affect the CNS and PNS. Pelizaeus-Merzbacher disease (PMD) is a myelin disorder of the CNS that arises from both mutational mechanisms. Investigating the molecular basis of PMD in patients and animal models is furthering our understanding of the disease, dosage sensitivity and proteolipid protein function during myelinogenesis.

Frameshift mutation in GJA12 leading to nystagmus, spastic ataxia and CNS dys-/demyelination

Mutations in GJA12 have been shown to cause Pelizaeus–Merzbacher-like disease (PMLD). We present two additional patients from one family carrying a homozygous frameshift mutation in GJA12. Both presented initially with nystagmus. The older girl developed ataxia first, then progressive spastic ataxia. The younger boy suffered from severe sensory neuropathy. Magnetic resonance imaging (MRI) of both children showed progressive demyelination in addition to dysmyelination, and also characteristic brainstem abnormalities. In children with nystagmus, ataxia and dysmyelination, mutation analysis of GJA12 should be considered early, especially if inheritance is autosomal recessive.

X inactivation phenotype in carriers of Pelizaeus-Merzbacher disease: Skewed in carriers of a duplication and random in carriers of point mutations

Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive disease caused by coding sequence mutations in the PLP gene, sub-microscopic duplications of variable sizes including the PLP gene or very rarely deletions of the PLP gene. We analysed the X inactivation pattern in blood of PMD female carriers with duplications and with point mutations. In the majority of duplication carriers (7/11), the X chromosome bearing the duplication was preferentially inactivated, whereas a random pattern of X inactivation was detected in point mutation carriers (3/3), a deletion carrier (1/1), affected females (4/4) who did not have a recognised mutation and normal control females. However 2/5 non-carrier female relatives of patients with a duplication, had skewed X inactivation. The skewed pattern of inactivation observed in most duplication carriers and not in mutation carriers suggests a) that there is selection against those cells in which the duplicated X chromosome is active and b) other expressed sequences within the duplicated region rather than mutant PLP may be responsible. Since the skewed X inactivation did not segregate with the disease in two families and the pattern of X inactivation was variable among the duplication carriers, the pattern X inactivation is an unsuitable diagnostic tool for female carriers of PMD.

Comparative mapping of 50 human chromosome 9 loci in the laboratory mouse

We have set out to produce a comprehensive comparative map between human chromosome 9 (HSA9) and the laboratory mouse. The mouse homologues of 50 loci that were known to map to HSA9 were mapped by interspecific backcross linkage analysis. Ten loci from the short arm of HSA9 were mapped, and 40 from HSA9q, with 24 markers coming from the HSA9q33-q34 region--a part of the chromosome known to be very gene rich. Fifteen new assignments have been made--Ak3, Ctsl, Cntfr, C8g, D2H9S46E, Eng, Gcnt1, Irebp, Pappa, Ptgds, Snf212, Tal2, Tmod, Vav2, and Vldlr, the human homologues of which all map to HSA9. In addition, the assignment of Snf212 and Vldlr to MMU19 has defined a new region of synteny between the proximal portion of the short arm of HSA9 and the mouse.

Complex chromosomal rearrangement and associated counseling issues in a family with Pelizaeus-Merzbacher disease.

We report cytogenetic and molecular findings in a family in which Pelizaeus‐Merzbacher disease has arisen by a sub‐microscopic duplication of the proteolipid protein (PLP1) gene involving the insertion of ∼600 kb from Xq22 into Xq26.3. The duplication arose in an asymptomatic mother on a paternally derived X chromosome and was inherited by her son, the proband, who is affected with Pelizaeus‐Merzbacher disease. The mother also carries a large interstitial deletion of ∼70 Mb extending from Xq21.1 to Xq27.3, which is present in a mosaic form. In lymphocytes, the mother has no normal cells, having one population with three copies of the PLP1gene (one normal X and one duplication X chromosome) and the other population having only one copy of the PLP1 gene (one normal X and one deleted X chromosome). Her karyotype is 46,XX.ish dup (X) (Xpter → Xq26.3::Xq22 → Xq22::Xq26.3 → Xqter)(PLP++)/46,X,del(X)(q21.1q27.3).ish del(X)(q21.1q27.3)(PLP−). Both ends of the deletion have been mapped by fluorescence in situ hybridization using selected DNA clones and neither involves the PLP1 gene or are in the vicinity of the duplication breakpoints. Prenatal diagnosis was carried out in a recent pregnancy and the complex counseling issues associated with these chromosomal rearrangements are discussed.