Okihiro syndrome refers to the association of forearm malformations with Duane syndrome of eye retraction. Based on the reported literature experience, clinical diagnosis of the syndrome can be elusive, owing to the variable presentation in families reported. Specifically, there is overlap of clinical features with other conditions, most notably Holt-Oram syndrome, a condition resulting from mutation of the TBX5 locus and Townes-Brocks syndrome, known to be caused by mutations in the SALL1 gene. Arising from our observation of several malformations in Okihiro syndrome patients which are also described in Townes-Brocks syndrome, we postulated that Okihiro syndrome might result from mutation of another member of the human SALL gene family. We have characterized the human SALL4 gene on chromosome 20q13.13-q13.2. Moreover, we have identified literature reports of forelimb malformations in patients with cytogenetically identifiable abnormalities of this region. We here present evidence in 5 of 8 affected families that mutation at this locus results in the Okihiro syndrome phenotype.

SALL4 is one out of four human homologues of the Drosophila region-specific homeotic gene spalt(sal). Heterozygous mutations of SALL4 on chromosome 20q13.13→ q13.2 cause the autosomal dominant Okihiro syndrome which is characterized by radial limb defects, Duane anomaly and hearing loss. We have partially cloned the murine homologue of this gene, named Sall4, and completed the coding sequence by comparison to available EST and genomic sequences in the GenBank database. This comparison also revealed the chromosomal location of Sall4 on mouse chromosome 2H3 and suggested that a predicted testis expressed gene Tex20 at the very same locus is most likely not a gene on its own but part of the Sall4 3′ UTR. We analyzed the expression of Sall4 during early embryogenesis by whole mount in situ hybridization and in the adult mouse by Northern blotting. In adult tissues, Sall4 expression is only found in testis and ovary. During embryonic development, Sall4 expression is widespread in early embryos and becomes gradually confined to the head region and the primitive streak. Prominent expression in the developing midbrain, branchial arches and the limbs suggests an important function of Sall4 during development of these structures as expected from the observation in Okihiro syndrome patients.

BACKGROUND: Partial deletions of the AZFc region of the Y chromosome were reported to be a significant risk factor for oligo-/azoospermia. In this study, we assessed the occurrence and frequency of partial AZFc microdeletions in patients with spermatogenic failure and in controls with normal spermatogenesis. METHODS: In a retrospective study design, gr/gr, b1/b3 and b2/b3 deletions were analysed by multiplex PCR in 170 men with normal spermatogenesis and 348 men with non-obstructive oligo-/azoospermia. RESULTS: gr/gr deletions were found in 14 men with oligozoospermia or azoospermia (4.0%) and in three normozoospermic men (1.8%) (NS). b1/b3 deletions were found both in controls (n 5 1) and in patients (n 5 1). b2/b3 deletions were significantly more frequent in the normozoospermic (five out of 170) than in the oligo-/azoospermic men (two out of 348). Three novel partial AZFc deletion patterns were found in four oligo-/azoospermic men. No correlation with semen or other clinical parameters was found. CONCLUSIONS: The frequency of gr/gr deletions is not significantly increased in men with oligo-/azoospermia, indicating that they are not sufficient per se to cause spermatogenetic impairment and infertility. b1/b3 and b2/b3 deletions are probably irrelevant for spermatogenesis. Novel deletion patterns found exclusively in infertile men suggest that other, still unexplored partial deletions might contribute to spermatogenic failure.

To test for human population substructure and to investigate human population history we have analysed Y-chromosome diversity using seven microsatellites (Y-STRs) and ten binary markers (Y-SNPs) in samples from eight regionally distributed populations from Poland (n = 913) and 11 from Germany (n = 1,215). Based on data from both Y-chromosome marker systems, which we found to be highly correlated (r = 0.96), and using spatial analysis of the molecular variance (SAMOVA), we revealed statistically significant support for two groups of populations: (1) all Polish populations and (2) all German populations. By means of analysis of the molecular variance (AMOVA) we observed a large and statistically significant proportion of 14% (for Y-SNPs) and 15% (for Y-STRs) of the respective total genetic variation being explained between both countries. The same population differentiation was detected using Monmonier's algorithm, with a resulting genetic border between Poland and Germany that closely resembles the course of the political border between both countries. The observed genetic differentiation was mainly, but not exclusively, due to the frequency distribution of two Y-SNP haplogroups and their associated Y-STR haplotypes: R1a1*, most frequent in Poland, and R1*(xR1a1), most frequent in Germany. We suggest here that the pronounced population differentiation between the two geographically neighbouring countries, Poland and Germany, is the consequence of very recent events in human population history, namely the forced human resettlement of many millions of Germans and Poles during and, especially, shortly after World War II. In addition, our findings have consequences for the forensic application of Y-chromosome markers, strongly supporting the implementation of population substructure into forensic Y chromosome databases, and also for genetic association studies.

Recently, several authors described the observation that RNA degradation does not correlate with the postmortem interval (PMI), but rather with other parameters like environmental impact and the circumstances of death. Therefore, the question arose if the analysis of gene expression could be a valuable tool in forensic genetics to contribute to the determination of the cause of death. In our study, six human tissues obtained from six individuals with PMI varying between 15 and 118 h were used for total RNA extraction. Quantification was performed using a GAPDH real-time assay, and the quality of mRNA was checked by amplification of different fragment lengths of the GAPDH transcript. In our set of samples, nearly all tissues in all PMI revealed satisfactory results, while skeletal muscle, followed by brain and heart, gave the best results. No correlation between PMI and RNA degradation could be detected, as very good results were observed for all tissues from the individual with the longest PMI. The highly promising results obtained in this study raise hopes that in the near future several fields of forensic investigation may profit from additional information about gene expression patterns and their correlation with pathological findings.

Gene expression analyses based on messenger RNA (mRNA) profiling require accurate data normalisation. When using endogenous reference genes, these have to be validated carefully. Therefore, we examined the transcript stability of 10 potential reference genes using quantitative real-time polymerase chain reaction: beta actin, 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase, TATA box-binding protein, hypoxanthine phosphoribosyl-transferase I, beta-2-microglobulin, hydroxymethylbilane synthase, succinate dehydrogenase complex, subunit A, cyclophilin A and ubiquitin C. The aim of the current study was to assess which reference genes show stable mRNA levels in human post mortem cardiac muscle, skeletal muscle and brain tissue. Considering cardiac muscle tissue, CYCA and TBP were identified as the most stable while in skeletal muscle tissue, SDHA and TBP, and in brain tissue, SDHA and HMBS turned out to be the most stable. Furthermore, we recommend a minimum of four carefully validated endogenous control genes for reliable data normalisation in human post mortem tissue. Parameters influencing the stability of transcript amounts were found to be mainly the post mortem interval in cardiac muscle and skeletal muscle tissue and the donor's cause of death in skeletal muscle and brain samples. Further parameters like gender, age at death and body mass index were found to influence mRNA quantities in skeletal muscle only. The set of stable control genes identified in this study may be used in further studies if the composition of the samples is similar to the one used here.

In the course of routine genotyping of forensic reference samples by multiplex PCR, an allelic drop-out due to mutations in the primer binding regions of the highly polymorphic STR marker ACTBP2 was observed in 17 samples. The variation rate was estimated to be 0.0014 (95% confidence interval: 0.0006-0.003). The most frequently found mutation was an G to A transition in the reverse primer binding region which was present in 14 out of 17 cases. To overcome the problem we have added a modified reverse primer to different multiplex kits that led to the correct genotype.

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