Background Holoprosencephaly (HPE) is the most common structural malformation of the human forebrain. There are several important HPE mutational target genes, including the transcription factor SIX3, which encodes an early regulator of Shh, Wnt, Bmp and Nodal signalling expressed in the developing forebrain and eyes of all vertebrates. Objective To characterise genetic and clinical findings in patients with SIX3 mutations. Methods Patients with HPE and their family members were tested for mutations in HPE-associated genes and the genetic and clinical findings, including those for additional cases found in the literature, were analysed. The results were correlated with a mutation-specific functional assay in zebrafish. Results In a cohort of patients (n = 800) with HPE, SIX3 mutations were found in 4.7% of probands and additional cases were found through testing of relatives. In total, 138 cases of HPE were identified, 59 of whom had not previously been clinically presented. Mutations in SIX3 result in more severe HPE than in other cases of non-chromosomal, non-syndromic HPE. An over-representation of severe HPE was found in patients whose mutations confer greater loss of function, as measured by the functional zebrafish assay. The gender ratio in this combined set of patients was 1.5:1 (F:M) and maternal inheritance was almost twice as common as paternal. About 14% of SIX3 mutations in probands occur de novo. There is a wide intrafamilial clinical range of features and classical penetrance is estimated to be at least 62%. Conclusions Our data suggest that SIX3 mutations result in relatively severe HPE and that there is a genotype–phenotype correlation, as shown by functional studies using animal models.

From a series of 107 females with Rett syndrome (RTT), we describe the long-term history of ten females with a deletion in the C-terminus of the MECP2 gene. We observed that their disorder profile is clinically recognizable with time and different from other atypical and milder RTT phenotypes. In females with hot spot deletions in the C-terminus, dystonia is present from childhood and results in a serious spine deformation in spite of preventive measures. Their adaptive behavior is surprisingly better preserved and in contrast with the typical decline in motor functioning. The delineation of disorder profiles by long-term clinical observation can teach us about genotype/phenotype relationships and eventually about the effect of epigenetic phenomena on the final phenotype.

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Mutations of the ZIC2 transcription factor gene are among the most common heterozygous variations detected in holoprosencephaly (HPE) patients, a patient group who lack critical midline forebrain specification due to defective embryonic signaling during development. Recent studies indicate that complete deficiency of the related murine Zic2 transcription factor can also be a contributing factor to variable midline deficiencies, presenting during mid-gastrulation, that could explain similar forebrain anomalies in this model system. Here we collect and summarize all available mutations in the human ZIC2 gene detected in HPE patients (21 published and 62 novel). Our analysis corroborates this mechanism proposed in mice by predicting loss-offunction as the likely pathogenetic mechanism common to most, if not all, of these mutations in HPE.

Cystic fibrosis (CF) is the first monogenic disorder for which single cell preimplantation genetic diagnosis (PGD) has been successfully applied. The spectrum of mutations in CF is extremely heterogeneous, and hence, the development of mutation-specific PGD protocols is impracticable. The current study reports the development and evaluation of a general multiplex marker polymerase chain reaction (PCR) protocol for PGD of CF. Four closely linked highly polymorphic (CA)(n) repeat markers D7S523, D7S486, D7S480 and D7S490, flanking the cystic fibrosis transmembrane regulator (CFTR) gene, were used. In 99% of the single cells tested (100 leukocytes and 50 blastomeres), multiplex PCR results were obtained and the overall allelic drop out (ADO) rate varied from 2 to 5%. After validation for the presence of ADO and additional alleles, 95% of the multiplex PCR results were accepted to construct the marker genotypes. Depending on the genotype of the couple, and taking into account the embryos lost for transfer due to validation criteria (5%), ADO (0-2%) and single recombination (1.1-3%), in general >90% of the embryos could be reliably genotyped by PGD using a single blastomere. The risk of misdiagnosis equals the chance of a double recombination between informative flanking markers and is <0.05%. Therefore, this polymorphic and multi-allelic marker system is a reliable and generally applicable alternative for mutation-directed PGD protocols. Furthermore, it provides a test for the origin of the detected genotype and also gives an indication of the chromosomal ploidy status of the blastomere tested.