Progress in the autosomal segmental aneusomy syndromes (SASs): single or multi-locus disorders?

Based on cytogenetic observations, several syndromes have been previously identified as microdeletion-based disorders. In this review, recent progress is presented regarding whether one or multiple genes can be implicated in the pathogenesis of these segmentally aneusomic syndromes. The syndromes discussed include Angelman, Alagille, Williams, Langer-Giedeon, Prader-Willi, Smith-Magenis, Miller-Dieker, and DiGeorge/velocardiofacial or the 22q11 deletion syndromes. For Angelman and Alagille syndromes, single genes have been identified, whereas for Williams and Langer-Giedion syndromes, more than one gene can be implicated. Although there has been significant progress in dissecting the molecular basis for the other disorders, the ultimate answer regarding one versus several genes remains to be determined.      

Missense mutation in a von Willebrand factor type A domain of the alpha 3(VI) collagen gene (COL6A3) in a family with Bethlem myopathy

The Bethlem myopathy is a rare autosomal dominant proximal myopathy characterized by early childhood onset and joint contractures. Evidence for linkage and genetic heterogeneity has been established, with the majority of families linked to 21q22.3 and one large family linked to 2q37, implicating the three type VI collagen subunit genes, COL6A1 (chromosome 21), COL6A2 (chromosome 21) and COL6A3 (chromosome 2) as candidate genes. Mutations of the invariant glycine residues in the triple-helical domain-coding region of COL6A1 and COL6A2 have been reported previously in the chromosome 21-linked families. We report here the identification of a G-->A mutation in the N-terminal globular domain-coding region of COL6A3 in a large American pedigree (19 affected, 12 unaffected), leading to the substitution of glycine by glutamic acid in the N2 motif, which is homologous to the type A domains of the von Willebrand factor. This mutation segregated to all affected family members, to no unaffected family members, and was not identified in 338 unrelated Caucasian control chromosomes. Thus mutations in either the triple-helical domain or the globular domain of type VI collagen appear to cause Bethlem myopathy.      

doublecortin is the major gene causing X-linked subcortical laminar heterotopia (SCLH)

Subcortical laminar heterotopia (SCLH), or 'double cortex', is a cortical dysgenesis disorder associated with a defect in neuronal migration. Clinical manifestations are epilepsy and mental retardation. This disorder, which mainly affects females, can be inherited in a single pedigree with lissencephaly, a more severe disease which affects the male individuals. This clinical entity has been described as X- SCLH/LIS syndrome. Recently we have demonstrated that the doublecortin gene, which is localized on the X chromosome, is implicated in this disorder. We have now performed a systematic mutation analysis of doublecortin in 11 unrelated females with SCLH (one familial and 10 sporadic cases) and have identified mutations in 10/11 cases. The sequence differences include nonsense, splice site and missense mutations and these were found throughout the gene. These results provide strong evidence that loss of function of doublecortin is the major cause of SCLH. The absence of phenotype-genotype correlations suggests that X-inactivation patterns of neuronal precursor cells are likely to contribute to the variable clinical severity of this disorder in females.      

Interchromosomal duplications of the adrenoleukodystrophy locus: a phenomenon of pericentromeric plasticity

A 9.7 kb segment encompassing exons 7-10 of the adrenoleukodystrophy (ALD) locus of the X chromosome has duplicated to specific locations near the pericentromeric regions of human chromosomes 2p11,10p11, 16p11 and 22q11. Comparative sequence analysis reveals 92-96% nucleotide identity, indicating that the autosomal ALD paralogs arose relatively recently during the course of higher primate evolution (5-10 million years ago). Analysis of sequences flanking the duplication region identifies the presence of an unusual GCTTTTTGC repeat which may be a sequence-specific integration site for the process of pericentromeric- directed transposition. The breakpoint sequence and phylogenetic analysis predict a two-step transposition model, in which a duplication from Xq28 to pericentromeric 2p11 occurred once, followed by a rapid distribution of a larger duplicon cassette among the pericentromeric regions. In addition to facilitating more effective mutation detection among ALD patients, these findings provide further insight into the molecular basis underlying a pericentromeric-directed mechanism for non- homologous interchromosomal exchange.      

Human mini-chromosomes in mouse embryonal stem cells

We have introduced human mini-chromosomes of 4 Mb and approximately 15 Mb in size into mouse embryonal stem cells. Although these human mini- chromosomes are stable in hamster and chicken cells, they re-arrange or segregate aberrantly in the embryonal stem cells and are rapidly lost in the absence of selection. However, one of the mini-chromosomes re- arranged, acquired mouse centromeric sequences and was then stably maintained for at least 60 population doublings in culture. This mini- chromosome, which is 4 Mb in size, is a candidate for a mouse germ line chromosome vector.      

A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy

Background  

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenic mutations setting the stage for successful gene therapy treatment. We have completed a study that directly deals with the following key issues that can be directly adapted to a gene therapy clinical trial using rAAV considering the following criteria: 1) A regional vascular delivery approach that will protect the patient from widespread dissemination of virus; 2) an approach to potentially facilitate safe passage of the virus for efficient skeletal muscle transduction; 3) the use of viral doses to accommodate current limitations imposed by vector production methods; 4) and at the same time, achieve a clinically meaningful outcome by transducing multiple muscles in the lower limb to prolong ambulation.     

Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal: lack of genotype/phenotype correlations

It has previously been shown that, in the heterozygous state, mutations in the SOX9 gene cause campomelic dysplasia (CD) and the often associated autosomal XY sex reversal. In 12 CD patients, 10 novel mutations and one recurrent mutation were characterized in one SOX9 allele each, and in one case, no mutation was found. Four missense mutations are all located within the high mobility group (HMG) domain. They either reduce or abolish the DNA-binding ability of the mutant SOX9 proteins. Among the five nonsense and three frameshift mutations identified, two leave the C-terminal transactivation (TA) domain encompassing residues 402-509 of SOX9 partly or almost completely intact. When tested in cell transfection experiments, the recurrent nonsense mutation Y440X, found in two patients who survived for four and more than 9 years, respectively, exhibits some residual transactivation ability. In contrast, a frameshift mutation extending the protein by 70 residues at codon 507, found in a patient who died shortly after birth, showed no transactivation. This is apparently due to instability of the mutant SOX9 protein as demonstrated by Western blotting. Amino acid substitutions and nonsense mutations are found in patients with and without XY sex reversal, indicating that sex reversal in CD is subject to variable penetrance. Finally, none of 18 female patients with XY gonadal dysgenesis (Swyer syndrome) showed an altered SOX9 banding pattern in SSCP assays, providing evidence that SOX9 mutations do not usually result in XY sex reversal without skeletal malformations.      

Dynamic and static phantoms for evaluation of digital subtraction angiographic systems

Two types of phantoms were developed with which to evaluate the overall performance of digital subtraction angiography (DSA) systems. A dynamic phantom, called a "fish bone" phantom, consists of polyethylene tubes that simulate blood vessels with various lesions, such as stenoses, ulcers, and aneurysms. With this phantom, washout curves were obtained representing the relationship between iodine content and time. It will be useful for qualitative assessment of DSA images, evaluation of different image-processing schemes, and studies of blood flow analysis. A static phantom, called a "C-D" phantom, can be used for measurement of quantitative contrast-detail (C-D) diagrams and for daily monitoring of DSA systems. This was constructed of tubes of seven different diameters (2.15-0.28 mm) and 14 different concentrations of contrast medium (100%-1.1% Renografin-76 [meglumine and sodium diatrizoate]). The C-D diagrams were determined from an observer performance study using C-D phantom images obtained at four different DSA settings.     

Needle biopsy of renal allografts: comparison of two techniques

Two techniques for renal allograft biopsy were retrospectively evaluated to compare relative safety and efficacy. After ultrasound (US) localization of the kidney and biopsy with a hand-held 14-gauge cutting needle, an adequate specimen was obtained in 74 of 77 cases (96%). Major complications occurred in six of these 77 cases (8%). One hundred four biopsies were performed by using a smaller 18-gauge cutting needle with a spring-loaded biopsy "gun" and real-time US guidance. With this newer technique, specimens adequate for diagnosis were obtained in 99 biopsies (95%). There was a single major complication with this technique (1%). The 18-gauge needle with real-time US guidance yields comparably adequate specimens with a lower frequency of complications.