Pseudoachondroplasia and multiple epiphyseal dysplasia: a 7-year comprehensive analysis of the known disease genes identify novel and recurrent mutations and provides an accurate assessment of their relative contribution.

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are relatively common skeletal dysplasias resulting in short-limbed dwarfism, joint pain, and stiffness. PSACH and the largest proportion of autosomal dominant MED (AD-MED) results from mutations in cartilage oligomeric matrix protein (COMP); however, AD-MED is genetically heterogenous and can also result from mutations in matrilin-3 (MATN3) and type IX collagen (COL9A1, COL9A2, and COL9A3). In contrast, autosomal recessive MED (rMED) appears to result exclusively from mutations in sulphate transporter solute carrier family 26 (SLC26A2). The diagnosis of PSACH and MED can be difficult for the nonexpert due to various complications and similarities with other related diseases and often mutation analysis is requested to either confirm or exclude the diagnosis. Since 2003, the European Skeletal Dysplasia Network (ESDN) has used an on-line review system to efficiently diagnose cases referred to the network prior to mutation analysis. In this study, we present the molecular findings in 130 patients referred to ESDN, which includes the identification of novel and recurrent mutations in over 100 patients. Furthermore, this study provides the first indication of the relative contribution of each gene and confirms that they account for the majority of PSACH and MED.

Preparation and validation of the first WHO international genetic reference panel for Fragile X syndrome.

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by expansion of a trinucleotide (CGG)n repeat sequence in the 5' untranslated region of the FMR1 gene, resulting in promoter hypermethylation and suppression of FMR1 transcription. Additionally, pre-mutation alleles in carrier males and females may result in Fragile X tremor ataxia syndrome and primary ovarian insufficiency, respectively. Fragile X is one of the most commonly requested molecular genetic tests worldwide. Quality assessment schemes have identified a wide disparity in allele sizing between laboratories. It is therefore important that clinical laboratories have access to characterized reference materials (RMs) to aid accurate allele sizing and diagnosis. With this in mind, a panel of genotyping RMs for Fragile X syndrome has been developed, which should be stable over many years and available to all diagnostic laboratories. Immortalized cell lines were produced by Epstein-Barr virus transformation of lymphocytes from consenting patients. Genomic DNA was extracted in bulk and RM aliquots were freeze-dried in glass ampoules. Twenty-one laboratories from seventeen countries participated in a collaborative study to assess their suitability. Participants evaluated the samples (blinded, in triplicate) in their routine methods alongside in-house and commercial controls. The panel of five genomic DNA samples was endorsed by the European Society of Human Genetics and approved as an International Standard by the Expert Committee on Biological Standardization at the World Health Organization.

Y chromosome detection by Real Time PCR and pyrophosphorolysis-activated polymerisation using free fetal DNA isolated from maternal plasma.

OBJECTIVES: To validate the use of Real Time PCR, a widely used technique that can detect very low levels of Y chromosomal sequence, and to assess the use of a highly sensitive PCR technique, pyrophosphorolysis-activated polymerisation (PAP), for fetal sex determination using free fetal DNA (ffDNA). METHODS: The fetal sex was determined by Real Time PCR in 58 pregnancies using ffDNA isolated from maternal plasma. In parallel with the Real Time PCR experiments, the presence of Y chromosome sequence was also determined using PAP on 54 isolated ffDNA samples. RESULTS: Both techniques detected Y chromosome sequence at very low levels with 98% specificity and 100% sensitivity (Real Time n = 44, PAP n = 54). Furthermore, the PAP technique was shown to be more robust than the Real Time PCR as none of the samples tested failed to meet the acceptance criteria. Combining the two techniques for male fetal sex detection from maternal blood plasma increases the sensitivity and specificity to 100% in this series. CONCLUSIONS: This study shows that both Real Time PCR and PAP can be used for Y chromosome detection on ffDNA. Furthermore, by using PAP in combination with Real Time PCR more reliable early prenatal sexing can be performed using ffDNA.

Preselection of cases through expert clinical and radiological review significantly increases mutation detection rate in multiple epiphyseal dysplasia.

Skeletal dysplasias are difficult to diagnose for the nonexpert. In a previous study of patients with multiple epiphyseal dysplasia (MED), we identified cartilage oligomeric matrix protein (COMP) mutations in only 36% of cases and suspected that the low-mutation detection rate was partially due to misdiagnosis. We therefore instituted a clinical-radiographic review system, whereby all cases were evaluated by a panel of skeletal dysplasia experts (European Skeletal Dysplasia Network). Only those patients in whom the diagnosis of MED was confirmed by the panel were screened for mutations. Under this regimen the mutation detection rate increased to 81%. When clinical-radiological diagnostic criteria were relaxed the mutation rate dropped to 67%. We conclude that expert clinical-radiological review can significantly enhance mutation detection rates and should be part of any diagnostic mutation screening protocol for skeletal dysplasias.

Benchmark for evaluating the quality of DNA sequencing: proposal from an international external quality assessment scheme.

BACKGROUND: In the past 15 years, clinical laboratory science has been transformed by the use of technologies that cross the traditional boundaries between laboratory disciplines. However, during this period, issues of quality have not always been given adequate attention. The European Molecular Genetics Quality Network (EMQN) has developed a novel external quality assessment scheme for evaluation of DNA sequencing. We report the results of an international survey of the quality of DNA sequencing among 64 laboratories from 21 countries. METHODS: Current practice for DNA sequence analysis was established by use of an online questionnaire. Participating laboratories were provided with 4 DNA samples of validated genotype. Evaluation of the results included assessing the quality of sequence data, variant genotypes, and mutation nomenclature. To accommodate variations in mutation nomenclature, variants indicated by participants were scored for compliance with 3 acceptable marking schemes. RESULTS: A total of 346 genotypes were analyzed. Of these, 19 (5%) genotyping errors were made. Of these, 10 (53%) were false-negative and 9 (47%) were false-positive results. A further 27 (8%) errors were made in naming mutations. Results were analyzed for 3 indicators of data quality: PHRED quality scores, Quality Read Length, and Quality Read Overlap. Most laboratories produced results of acceptable diagnostic quality as judged by these indicators. The results were used to calculate a consensus benchmark for DNA sequencing against which individual laboratories could rank their performance. CONCLUSIONS: We propose that the consensus benchmark can be used as a baseline against which the aggregate and individual laboratory standard of DNA sequencing may be tracked from year to year.

Prenatal diagnosis in a family with X-linked hydrocephalus.

The neural cell adhesion molecule L1 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily of cell adhesion molecules (CAMs). Its expression is essential during embryonic development of the nervous system and it is involved in cognitive function and memory. Mutations in the L1CAM gene are responsible for four related L1 disorders; X-linked hydrocephalus/HSAS (Hydrocephalus as a result of Stenosis of the Aqueduct of Sylvius), MASA (Mental retardation, Aphasia, Shuffling gait, and Adducted thumbs) syndrome, X-linked complicated spastic paraplegia type I (SPG1) and X-linked Agenesis of the Corpus Callosum (ACC). These four disorders represent a clinical spectrum that varies both between and within families. The main clinical features of this spectrum are Corpus callosum hypoplasia, mental Retardation, Adducted thumbs, Spastic paraplegia and Hydrocephalus (CRASH syndrome). Since there is no biochemically assayed disease marker, molecular analysis of the L1CAM gene is the only means of confirming a clinical diagnosis. Most L1CAM mutations reported to date are point mutations (missense, nonsense, splice site) and only a few patients with larger rearrangements have been documented. We have characterised a rare intragenic deletion of the L1CAM gene in a sample of DNA extracted from a chorionic villus biopsy (CVB) performed at 12 weeks' gestation. =

Novel and recurrent mutations in the C-terminal domain of COMP cluster in two distinct regions and result in a spectrum of phenotypes within the pseudoachondroplasia -- multiple epiphyseal dysplasia disease group.

Pseudoachondroplasia (PSACH) and some forms of multiple epiphyseal dysplasia (MED) result from mutations in the gene encoding cartilage oligomeric matrix protein (COMP). COMP is a large pentameric glycoprotein found predominantly in the extracellular matrix of cartilage, tendon, and ligament. As a modular protein, it is composed of a coiled-coil domain, four type II (T2) repeats, eight type III (T3) repeats, and a large globular C-terminal domain (CTD). The majority (>85%) of COMP mutations causing PSACH or MED are found in the exons encoding the T3 repeats, and the disease mechanism has been characterised in detail. Much less is known about disease-causing mutations in the CTD; in 10 years only seven mutations have been identified. In this study, we describe eight novel and two recurrent mutations that we have recently identified in patients with PSACH or MED. Interestingly, these mutations result in a spectrum of disease, ranging from mild MED to severe PSACH. Mapping of all known COMP CTD mutations on a three-dimensional model of the C-terminal domain shows that the CTD mutations cluster in two distinct regions. These regions are probably important in stabilising the T3-CTD structure and mediating intra- or intermolecular interactions.

COMP mutation screening as an aid for the clinical diagnosis and counselling of patients with a suspected diagnosis of pseudoachondroplasia or multiple epiphyseal dysplasia.

The skeletal dysplasias are a clinically and genetically heterogeneous group of conditions affecting the development of the osseous skeleton and fall into the category of rare genetic diseases in which the diagnosis can be difficult for the nonexpert. Two such diseases are pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED), which result in varying degrees of short stature, joint pain and stiffness and often resulting in early onset osteoarthritis. PSACH and some forms of MED result from mutations in the cartilage oligomeric matrix protein (COMP) gene and to aid the clinical diagnosis and counselling of patients with a suspected diagnosis of PSACH or MED, we developed an efficient and accurate molecular diagnostic service for the COMP gene. In a 36-month period, 100 families were screened for a mutation in COMP and we identified disease-causing mutations in 78% of PSACH families and 36% of MED families. Furthermore, in several of these families, the identification of a disease-causing mutation provided information that was immediately used to direct reproductive decision-making.