Barbara Zoll

Genotyping in 46 patients with tentative diagnosis of Treacher Collins syndrome revealed unexpected phenotypic variation

To define the range of phenotypic expression in Treacher Collins syndrome (TCS; Franceschetti–Klein syndrome), we performed mutation analysis in the TCOF1 gene in 46 patients with tentative diagnosis of TCS and evaluated the clinical data, including a scoring system. A total of 27 coding exons of TCOF1 and adjacent splice junctions were analysed by direct sequencing. In 36 patients with a clinically unequivocal diagnosis of TCS, we detected 28 pathogenic mutations, including 25 novel alterations. No mutation was identified in the remaining eight patients with unequivocal diagnosis of TCS and 10 further patients, in whom the referring diagnosis of TCS was clinically doubtful. There is no overt genotype–phenotype correlation except that conductive deafness is significantly less frequent in patients with mutations in the 3′ part of the open reading frame. Inter- and intrafamilial variation is wide. Some mutation carriers, parents of typically affected patients, are so mildly affected that the diagnosis might be overlooked clinically. This suggests that modifying factors are important for phenotypic expression. Based on these findings, minimal diagnostic criteria were defined: downward slanting palpebral fissures and hypoplasia of the zygomatic arch. The difficulties in genetic counselling, especially diagnosis of family members with a mild phenotype, are described.

Distribution of epidermal growth factor receptor gene amplification in brain tumours and correlation to prognosis.

In 75 gliomas and 31 meningiomas, mutations at the epidermal growth factor receptor (EGFR) gene locus were restricted to gliomas. The ligands of this receptor, epidermal growth factor and transforming growth factor alpha, lacked quantitative changes at their loci in gliomas and meningiomas. EGFR gene amplification occurred in astrocytomas, oligodendrogliomas, ependymomas and glioblastomas. The frequency of this mutation significantly increased with the malignancy grade and the patients age. Especially in glioblastomas of individuals aged over 64 years, EGFR gene mutations were observed without chromosome-10-specific allele losses. This finding contradicts the hypothesis that deletion of one entire chromosome 10 regularly precedes EGFR gene amplification in primary glioblastomas of patients aged over 50 years. It was found that most individuals whose gliomas carry an EGFR gene mutation have a poor prognosis, comparable to that of glioblastoma patients even when the tumour is graded as benign.

Isolation and Characterization of the Human Forkhead Gene FOXQ1

We have isolated a human genomic and cDNA clone that encodes a protein of 403 amino acids and belongs to the family of the FOX transcription factors (previously called HNF-3/forkhead transcription factors). The 2.7-kb transcript of the human FOXQ1 gene is expressed predominantly in the stomach, trachea, bladder and salivary gland. Additionally, overexpression of human FOXQ1 was shown in colorectal adenocarcinoma and lung carcinoma cell lines. The FOXQ1 gene is located on chromosome 6p23-25. Databank analysis shows 82% homology with the mouse Foxq1 gene (formerly Hfh-1L) and with a revised sequence of the rat FoxQ1 gene (formerly HFH-1). The DNA-binding motif, named HNF-3/forkhead domain, is well conserved, showing 100% identity in human, mouse, and rat. The human protein sequence contains two putative transcriptional activation domains, which share a high amino acid identity with the corresponding mouse and rat domains.

Linkage data suggesting allelic heterogeneity for paramyotonia congenita and hyperkalemic periodic paralysis on chromosome 17

SummaryParamyotonia congenita (PC), an autosomal dominant non-progressive muscle disorder, is characterised by cold-induced stiffness followed by muscle weakness. The weakness is caused by a dysfunction of the sodium channel in muscle fibre. Parts of the gene coding for the α-subunit of the sodium channel of the adult human skeletal muscle (SCN4A) have been localised on chromosome 17. To investigate the role of this gene in the etiology of PC, a linkage analysis in 17 well-defined families was carried out. The results (z=20.61, Θ=0.001) show that the mutant gene responsible for the disorder is indeed tightly linked to the SCN4A gene. The mutation causing hyperkalemic periodic paralysis (HyperPP) with myotonia has previously been mapped to this gene locus by the same candidate gene approach. Thus, our data suggest that PC and HyperPP are caused by allelic mutations at a single locus on chromosome 17.

The molecular genetic analysis of haemophilia A; characterization of six partial deletions in the factor VIII gene

SummaryIn a survey of 528 unrelated haemophilia A patients, six partial deletions of the factor VIII (FVIII) gene were detected by Southern blotting. These deletions were further mapped by a combination of Southern blotting and polymerase chain reaction amplification and found to vary in length between 4.7kb and 57kb. The frequency of detectable FVIII gene deletions (about 1%) is thus considerably lower than previously reported. Statistical analysis of currently available data did not provide any evidence for a deletion “hotspot”. Four of the six deletion patients reported here possessed inhibitors. Taken together with previous data, deletion of the FVIII gene was found to be associated with an approximately fivefold higher risk of developing inhibitors compared with other severe haemophiliacs without gene deletions.

Comparison between the allelic frequency distribution of the Ha-ras 1 locus in normal individuals and patients with lymphoma, breast, and ovarian cancer

SummaryAnalysis of 426 alleles of the human Ha-ras 1 locus in DNA samples of cancer patients and controls reveals no obvious association between specific restriction fragments and a genetic predisposition to malignancy. However, differences in the allele distribution in leukocyte and tumour DNAs of cancer patients may be related to an involvement of the Ha-ras1 gene in tumorigenesis.

CHARGE and Kabuki syndromes: A phenotypic and molecular link

CHARGE syndrome is a complex developmental disorder caused by mutations in the chromodomain helicase DNA-binding gene CHD7. Kabuki syndrome, another developmental disorder, is characterized by typical facial features in combination with developmental delay, short stature, prominent digit pads and visceral abnormalities. Mutations in the KMT2D gene, which encodes a H3K4 histone methyltransferase, are the major cause of Kabuki syndrome. Here, we report a patient, who was initially diagnosed with CHARGE syndrome based on the spectrum of inner organ malformations like choanal hypoplasia, heart defect, anal atresia, vision problems and conductive hearing impairment. While sequencing and MLPA analysis of all coding exons of CHD7 revealed no pathogenic mutation, sequence analysis of the KMT2D gene identified the heterozygous de novo nonsense mutation c.5263C > T (p.Gln1755*). Thus, our patient was diagnosed with Kabuki syndrome. By using co-immunoprecipitation, immunohistochemistry and direct yeast two hybrid assays, we could show that, like KMT2D, CHD7 interacts with members of the WAR complex, namely WDR5, ASH2L and RbBP5. We therefore propose that CHD7 and KMT2D function in the same chromatin modification machinery, thus pointing out a mechanistic connection, and presenting a probable explanation for the phenotypic overlap between Kabuki and CHARGE syndromes.

Mutation Update for Kabuki Syndrome Genes KMT2D and KDM6A and Further Delineation of X-Linked Kabuki Syndrome Subtype 2.

Kabuki syndrome (KS) is a rare but recognizable condition that consists of a characteristic face, short stature, various organ malformations, and a variable degree of intellectual disability. Mutations in KMT2D have been identified as the main cause for KS, whereas mutations in KDM6A are a much less frequent cause. Here, we report a mutation screening in a case series of 347 unpublished patients, in which we identified 12 novel KDM6A mutations (KS type 2) and 208 mutations in KMT2D (KS type 1), 132 of them novel. Two of the KDM6A mutations were maternally inherited and nine were shown to be de novo. We give an up‐to‐date overview of all published mutations for the two KS genes and point out possible mutation hot spots and strategies for molecular genetic testing. We also report the clinical details for 11 patients with KS type 2, summarize the published clinical information, specifically with a focus on the less well‐defined X‐linked KS type 2, and comment on phenotype–genotype correlations as well as sex‐specific phenotypic differences. Finally, we also discuss a possible role of KDM6A in Kabuki‐like Turner syndrome and report a mutation screening of KDM6C (UTY) in male KS patients.

Cytogenetic investigation of 103 patients with primary or secondary amenorrhea

SummaryCytogenetic investigations were carried out on 103 women presenting with either primary (n=88) or secondary (n=15) amenorrhea. A sex chromosome anomaly was found in 26% and 33% of these patients, respectively.Other studies on women with primary amenorrhea have found a similar or even higher percentage of patients with an abnormal karyotype. It is therefore suggested that all women with absence of menstruation after the age of 16 years should be investigated cytogenetically.The surprisingly high percentage of pathological karyotypes among the secondary amenorrhea group does indicate that sex chromosome anomalies cannot be ruled out in women who have had apparently normal ovarian function for at least some time, and therefore more patients from this group should be selected for chromosome analysis.

Molecular and genetic characterisation of German families with paramyotonia congenita and demonstration of founder effect in the Ravensberg families

Eighteen German families with a history of paramyotonia congenita (PC) were characterised by genetic und mutational analysis at the SCN4A locus, which encodes the α-subunit of the adult skeletal muscle sodium channel. We concentrated our analysis primarily on these families to test the hypothesis that a predominance of one common mutation occurs in all German PC families and that this mutation arose in a common ancestor originating in the North-West of the country. The present eighteen PC families exhibit two different mutations (R1448C and R1448H) on various SCN4A dinucleotide repeat haplotypes and therefore the majority of the mutations probably occurred independently. However, the R1448H mutation is extremely frequent in the North-West of Germany (Ravensberger Land) on a specific SCN4A microsatellite haplotype, indicating a founder effect within this subpopulation. Our results suggest that the R1448C/R1448H mutations are by far the most common to be associated with the PC phenotype in the German population.