Huntington's disease (HD) is an incurable neuropsychiatric disease associated with CAG repeat expansion within a widely expressed gene that causes selective neuronal death. To understand its normal function, we have created a targeted disruption in exon 5 of Hdh (Hdhex5), the murine homolog of the HD gene. Homozygotes die before embryonic day 8.5, initiate gastrulation, but do not proceed to the formation of somites or to organogenesis. Mice heterozygous for the Hdhex5 mutation display increased motor activity and cognitive deficits. Neuropathological assessment of two heterozygous mice shows significant neuronal loss in the subthalamic nucleus. These studies show that the HD gene is essential for postimplantation development and that it may play an important role in normal functioning of the basal ganglia.

Familial exudative vitreoretinopathy (FEVR) is a hereditary ocular disorder characterized by a failure of peripheral retinal vascularization. Loci associated with FEVR map to 11q13-q23 (EVR1; OMIM 133780, ref. 1), Xp11.4 (EVR2; OMIM 305390, ref. 2) and 11p13-12 (EVR3; OMIM 605750, ref. 3). Here we have confirmed linkage to the 11q13-23 locus for autosomal dominant FEVR in one large multigenerational family and refined the disease locus to a genomic region spanning 1.55 Mb. Mutations in FZD4, encoding the putative Wnt receptor frizzled-4, segregated completely with affected individuals in the family and were detected in affected individuals from an additional unrelated family, but not in normal controls. FZD genes encode Wnt receptors, which are implicated in development and carcinogenesis. Injection of wildtype and mutated FZD4 into Xenopus laevis embryos revealed that wildtype, but not mutant, frizzled-4 activated calcium/calmodulin-dependent protein kinase II (CAMKII) and protein kinase C (PKC), components of the Wnt/Ca(2+) signaling pathway. In one of the mutants, altered subcellular trafficking led to defective signaling. These findings support a function for frizzled-4 in retinal angiogenesis and establish the first association between a Wnt receptor and human disease.

CAG trinucleotide expansion is the molecular basis of Huntington's disease worldwide and is a highly sensitive and specific marker for inheritance of the disease mutation.

Huntington disease is associated with an unstable and expanded (CAG) trinucleotide repeat. We have analysed the CAG expansion in different tissues from 12 affected individuals. All tissues examined were found to display some repeat mosaicism, with the greatest levels detected in brain and sperm. Regions within the brain showing most obvious neuropathology, such as the basal ganglia and the cerebral cortex, displayed the greatest mosaicism, whereas the cerebellar cortex, which is seldom involved, displayed the lowest degree of CAG instability. In two cases of childhood onset disease we detected differences of 8 and 13 trinucleotides between the cerebellum and other regions of the brain. Our results provide evidence for tissue specific instability of the CAG repeat, with the largest CAG repeat lengths in affected regions of the brain.

Deletions of the DAZ gene family in distal Yq11 are always associated with deletions of the azoospermia factor c (AZFc) region, which we now estimate extends to 4.94 Mb. Because more Y gene families are located in this chromosomal region, and are expressed like the DAZ gene family only in the male germ line, the testicular pathology associated with complete AZFc deletions cannot predict the functional contribution of the DAZ gene family to human spermatogenesis. We therefore established a DAZ gene copy specific deletion analysis based on the DAZ-BAC sequences in GenBank. It includes the deletion analysis of eight DAZ-DNA PCR markers [six DAZ-single nucleotide varients (SNVs) and two DAZ-sequence tag sites (STS)] selected from the 5' to the 3'end of each DAZ gene and a deletion analysis of the gene copy specific EcoRV and TaqI restriction fragments identified in the internal repetitive DAZ gene regions (DYS1 locus). With these diagnostic tools, 63 DNA samples from men with idiopathic oligozoospermia and 107 DNA samples from men with proven fertility were analysed for the presence of the complete DAZ gene locus, encompassing the four DAZ gene copies. In five oligozoospermic patients, we found a DAZ-SNV/STS and DYS1/EcoRV and TaqI fragment deletion pattern indicative for deletion of the DAZ1 and DAZ2 gene copies; one of these deletions could be identified as a 'de-novo' deletion because it was absent in the DAZ locus of the patient's father. The same DAZ deletions were not found in any of the 107 fertile control samples. We therefore conclude that the deletion of the DAZ1/DAZ2 gene doublet in five out of our 63 oligozoospermic patients (8%) is responsible for the patients' reduced sperm numbers. It is most likely caused by intrachromosomal recombination events between two long repetitive sequence blocks (AZFc-Rep1) flanking the DAZ gene structures.

We have directly compared intergenerational stability of intermediate alleles (IAs) derived from new mutation families (IANM) for Huntington disease (HD) with IAs in the general population (IAGP) which occur in approximately 1 in 50 persons. Analysis of meiotic events in blood and sperm reveals that IANM are significantly more unstable than IAGP despite similar size. However, for both IANM and IAGP CAG changes were small and risks for inheriting an expansion into the HD affected range were low. Sequence analysis reveals that the CAG tract is generally interrupted by a penultimate CAA in IAGP, IANM and alleles in the affected range. In one new mutation family, however, two A-->G mutations result in a pure CAG tract which is associated with very marked instability. These mutations alter the predicted DNA hairpin structure with a predicted increase in the likelihood of large expansion, supporting the model that hairpin loop formation plays an important role in trinucleotide instability.

The polymorphic CAG repeat that is expanded on Huntington disease (HD) chromosomes is flanked by a CCG repeat. Here we show that this CCG tract, previously assumed to be invariant at seven CCG repeats, is also polymorphic. We have identified five CCG alleles from 205 normal chromosomes, with 137 (67%) having alleles of seven repeats, five (2%) with nine repeats, 61 (30%) with 10 repeats, one (0.5%) with 11 repeats and one (0.5%) with 12 repeats. In contrast, analysis of 113 HD chromosomes revealed that the majority (105 chromosomes, 93%) contained seven CCG repeats, while the remaining eight chromosomes (7%) had allele sizes of 10 CCG repeats. Despite evidence that both CAG and CCG are polymorphic on normal chromosomes, we have found that it is only the CAG length that has a significant impact on age of onset. The discovery of larger sized CCG alleles, however, has significant implications for the assessment of CAG repeat length, particularly for persons with estimated CAG size of 36-42 repeats, since an overestimation of CAG length in this range could result in erroneous information being imparted to patients.

The stabilization of peptide secondary structure via stapling is a ubiquitous goal for creating new probes, imaging agents, and drugs. Inspired by indole‐derived crosslinks found in natural peptide toxins, we employed ortho‐phthalaldehydes to create isoindole staples, thus transforming inactive linear and monocyclic precursors into bioactive monocyclic and bicyclic products. Mild, metal‐free conditions give an array of macrocyclic α‐melanocyte‐stimulating hormone (α‐MSH) derivatives, of which several isoindole‐stapled α‐MSH analogues (Ki≈1 nm) are found to be as potent as α‐MSH. Analogously, late‐stage intra‐annular isoindole stapling furnished a bicyclic peptide mimic of α‐amanitin that is cytotoxic to CHO cells (IC50=70 μm). Given its user‐friendliness, we have termed this approach FlICk (fluorescent isoindole crosslink) chemistry.