Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS or SACS) is a neurodegenerative disease frequent in northeastern Québec. In a previous study, we localized the disease gene to chromosome region 13q11 by identifying excess sharing of a marker allele in patients followed by linkage analysis and haplotyping. To create a detailed physical map of this region, we screened CEPH mega-YACs with 41 chromosome 13 sequence-tagged-sites (STSs) known to map to 13q11-q12. The YAC contig, composed of 27 clones, extends on the genetic map from D13S175 to D13S221, an estimated distance of at least 19.3 cM. A high-resolution BAC and PAC map that includes the ARSACS critical region flanked by D13S1275 and D13S292 was constructed. These YAC and BAC/PAC maps allowed the accurate placement of 29 genes and ESTs previously mapped to the proximal region of chromosome 13q. We confirmed the position of two candidate genes within the critical region and mapped the other 27 genes and ESTs to nearby intervals. Six BAC/PAC clones form a contig between D13S232 and D13S787 for sequencing within the ARSACS critical region.

In humans, deletion of any one of three Y-chromosomal regions—AZFa, AZFb or AZFc—disrupts spermatogenesis, causing infertility in otherwise healthy men1,2,3,4,5. Although candidate genes have been identified in all three regions3,6,7,8, no case of spermatogenic failure has been traced to a point mutation in a Y-linked gene, or to a deletion of a single Y-linked gene. We sequenced the AZFa region of the Y chromosome and identified two functional genes previously described: USP9Y (also known as DFFRY) and DBY (refs 7,8). Screening of the two genes in 576 infertile and 96 fertile men revealed several sequence variants, most of which appear to be heritable and of little functional consequence. We found one de novo mutation in USP9Y: a 4-bp deletion in a splice-donor site, causing an exon to be skipped and protein truncation. This mutation was present in a man with nonobstructive azoospermia (that is, no sperm was detected in semen), but absent in his fertile brother, suggesting that the USP9Y mutation caused spermatogenic failure. We also identified a single-gene deletion associated with spermatogenic failure, again involving USP9Y, by re-analysing a published study.

Campomelic dysplasia (CD), a skeletal malformation syndrome with or without XY sex reversal, is usually caused by mutations within the SOX9 gene on distal 17q. Several CD translocation and inversion cases have been described with breakpoints outside the coding region, mapping to locations >130 kb proximal to SOX9. Such cases are generally less severely affected than cases with SOX9 coding-region mutations, as is borne out by three new translocation cases that we present. We have cloned the region extending 1.2 Mb upstream of the SOX9 gene in overlapping bacterial-artificial-chromosome and P1-artificial-chromosome clones and have established a restriction map with rare-cutter enzymes. With sequence-tagged-site-content mapping in somatic-cell hybrids, as well as with FISH, we have precisely mapped the breakpoints of the three new and of three previously described CD cases. The six CD breakpoints map to an interval that is 140-950 kb proximal to the SOX9 gene. With exon trapping, we could isolate five potential exons from the YAC 946E12 that spans the region, four of which could be placed in the contig in the vicinity of the breakpoints. They show the same transcriptional orientation, but only two have an open reading frame (ORF). We failed to detect expression of these fragments in several human and mouse cDNA libraries, as well as on northern blots. Genomic sequence totaling 1,063 kb from the SOX9 5'-flanking region was determined and was analyzed by the gene-prediction program GENSCAN and by a search of dbEST and other databases. No genes or transcripts could be identified. Together, these data suggest that the chromosomal rearrangements most likely remove one or more cis-regulatory elements from an extended SOX9 control region.