Coffin-Siris syndrome and the BAF complex: genotype-phenotype study in 63 patients.

De novo germline variants in several components of the SWI/SNF-like BAF complex can cause Coffin-Siris syndrome (CSS), Nicolaides-Baraitser syndrome (NCBRS), and nonsyndromic intellectual disability. We screened 63 patients with a clinical diagnosis of CSS for these genes (ARID1A, ARID1B, SMARCA2, SMARCA4, SMARCB1, and SMARCE1) and identified pathogenic variants in 45 (71%) patients. We found a high proportion of variants in ARID1B (68%). All four pathogenic variants in ARID1A appeared to be mosaic. By using all variants from the Exome Variant Server as test data, we were able to classify variants in ARID1A, ARID1B, and SMARCB1 reliably as being pathogenic or nonpathogenic. For SMARCA2, SMARCA4, and SMARCE1 several variants in the EVS remained unclassified, underlining the importance of parental testing. We have entered all variant and clinical information in LOVD-powered databases to facilitate further genotype-phenotype correlations, as these will become increasingly important because of the uptake of targeted and untargeted next generation sequencing in diagnostics. The emerging phenotype-genotype correlation is that SMARCB1 patients have the most marked physical phenotype and severe cognitive and growth delay. The variability in phenotype seems most marked in ARID1A and ARID1B patients. Distal limbs anomalies are most marked in ARID1A patients and least in SMARCB1 patients. Numbers are small however, and larger series are needed to confirm this correlation.

MUTYH gene variants and breast cancer in a Dutch case­control study.

The MUTYH gene is involved in base excision repair. MUTYH mutations predispose to recessively inherited colorectal polyposis and cancer. Here, we evaluate an association with breast cancer (BC), following up our previous finding of an elevated BC frequency among Dutch bi-allelic MUTYH mutation carriers. A case­control study was performed comparing 1,469 incident BC patients (ORIGO cohort), 471 individuals displaying features suggesting a genetic predisposition for BC, but without a detectable BRCA1 or BRCA2 mutation (BRCAx cohort), and 1,666 controls. First, for 303 consecutive patients diagnosed before age 55 years and/or with multiple primary breast tumors, the MUTYH coding region and flanking introns were sequenced. The remaining subjects were genotyped for five coding variants, p.Tyr179Cys, p.Arg309Cys, p.Gly396Asp, p.Pro405Leu, and p.Ser515Phe, and four tagging SNPs, c.37-2487G>T, p.Val22Met, c.504+35G>A, and p.Gln338His. No bi-allelic pathogenic MUTYH mutations were identified. The pathogenic variant p.Gly396Asp and the variant of uncertain significance p.Arg309Cys occurred twice as frequently in BRCAx subjects as compared to incident BC patients and controls (p=0.13 and p=0.15, respectively). The likely benign variant p.Val22Met occurred less frequently in patients from the incident BC (p=0.03) and BRCAx groups (p=0.11), respectively, as compared to the controls. Minor allele genotypes of several MUTYH variants showed trends towards association with lobular BC histology. This extensive case­control study could not confirm previously reported associations of MUTYH variants with BC, although it was too small to exclude subtle effects on BC susceptibility.

Leiden Open Variation Database of the MUTYH gene.

The MUTYH gene encodes a DNA glycosylase involved in base excision repair (BER). Biallelic pathogenic MUTYH variants have been associated with colorectal polyposis and cancer. The pathogenicity of a few variants is beyond doubt, including c.536A4G/p.Tyr179Cys and c.1187G4A/p.Gly396Asp (previously c.494A4G/p.Tyr165Cys and c.1145G4A/p.Gly382Asp).However, for a substantial fraction of the detected variants, the clinical significance remains uncertain,compromising molecular diagnostics and thereby genetic counseling. We have established an interactive MUTYH gene sequence variant database (www.lovd.nl/MUTYH) with the aim of collecting and sharing MUTYH genotype and phenotype data worldwide. To support standard variant description, we chose NM_001128425.1 as the reference sequence. The database includes records with variants per individual, linked to available phenotype and geographic origin data as well as records with in vitro functional and in silico test data. As of April 2010, the database contains 1968 published and 423 unpublished submitted entries, and 230 and 61 unique variants,respectively. This open-access repository allows all involved to quickly share all variants encountered and communicate potential consequences, which will be especially useful to classify variants of uncertain significance.

Deep sequencing to reveal new variants in pooled DNA samples.

We evaluated massive parallel sequencing and long-range PCR (LRP) for rare variant detection and allele frequency estimation in pooled DNA samples. Exons 2 to 16 of the MUTYH gene were analyzed in breast cancer patients with Illumina's (Solexa) technology. From a pool of 287 genomic DNA samples we generated a single LRP product, while the same LRP was performed on 88 individual samples and the resulting products then pooled. Concentrations of constituent samples were measured with fluorimetry for genomic DNA and high-resolution melting curve analysis (HR-MCA) for LRP products. Illumina sequencing results were compared to Sanger sequencing data of individual samples. Correlation between allele frequencies detected by both methods was poor in the first pool, presumably because the genomic samples amplified unequally in the LRP, due to DNA quality variability. In contrast, allele frequencies correlated well in the second pool, in which all expected alleles at a frequency of 1% and higher were reliably detected, plus the majority of singletons (0.6% allele frequency). We describe custom bioinformatics and statistics to optimize detection of rare variants and to estimate required sequencing depth. Our results provide directions for designing high-throughput analyses of candidate genes.

Somatic loss of maternal chromosome 11 causes parent-of-origin-dependent inheritance in SDHD-linked paraganglioma and phaeochromocytoma families.

Germline mutations in succinate dehydrogenase subunits B, C and D (SDHB, SDHC and SDHD), genes encoding subunits of mitochondrial complex II, cause hereditary paragangliomas and phaeochromocytomas. In SDHB (1p36)- and SDHC (1q21)-linked families, disease inheritance is autosomal dominant. In SDHD (11q23)-linked families, the disease phenotype is expressed only upon paternal transmission of the mutation, consistent with maternal imprinting. However, SDHD shows biallelic expression in brain, kidney and lymphoid tissues (Baysal et al., 2000). Moreover, consistent loss of the wild-type (wt) maternal allele in SDHD-linked tumours suggests expression of the maternal SDHD allele in normal paraganglia. Here we demonstrate exclusive loss of the entire maternal chromosome 11 in SDHD-linked paragangliomas and phaeochromocytomas, suggesting that combined loss of the wt SDHD allele and maternal 11p region is essential for tumorigenesis. We hypothesize that this is driven by selective loss of one or more imprinted genes in the 11p15 region. In paternally, but not in maternally derived SDHD mutation carriers, this can be achieved by a single event, that is, non-disjunctional loss of the maternal chromosome 11. Thus, the exclusive paternal transmission of the disease can be explained by a somatic genetic mechanism targeting both the SDHD gene on 11q23 and a paternally imprinted gene on 11p15.5, rather than imprinting of SDHD.

A Novel Targeted Approach for Noninvasive Detection of Paternally Inherited Mutations in Maternal Plasma.

The challenge in noninvasive prenatal diagnosis for monogenic disorders lies in the detection of low levels of fetal variants in the excess of maternal cell-free plasma DNA. Next-generation sequencing, which is the main method used for noninvasive prenatal testing and diagnosis, can overcome this challenge. However, this method may not be accessible to all genetic laboratories. Moreover, shotgun next-generation sequencing as, for instance, currently applied for noninvasive fetal trisomy screening may not be suitable for the detection of inherited mutations. We have developed a sensitive, mutation-specific, and fast alternative for next-generation sequencing-mediated noninvasive prenatal diagnosis using a PCR-based method. For this proof-of-principle study, noninvasive fetal paternally inherited mutation detection was performed using cell-free DNA from maternal plasma. Preferential amplification of the paternally inherited allele was accomplished through a personalized approach using a blocking probe against maternal sequences in a high-resolution melting curve analysis-based assay. Enhanced detection of the fetal paternally inherited mutation was obtained for both an autosomal dominant and a recessive monogenic disorder by blocking the amplification of maternal sequences in maternal plasma.

High-resolution melting (HRM) re-analysis of a polyposis patients cohort reveals previously undetected heterozygous and mosaic APC gene mutations.

Familial adenomatous polyposis is most frequently caused by pathogenic variants in either the APC gene or the MUTYH gene. The detection rate of pathogenic variants depends on the severity of the phenotype and sensitivity of the screening method, including sensitivity for mosaic variants. For 171 patients with multiple colorectal polyps without previously detectable pathogenic variant, APC was reanalyzed in leukocyte DNA by one uniform technique: high-resolution melting (HRM) analysis. Serial dilution of heterozygous DNA resulted in a lowest detectable allelic fraction of 6% for the majority of variants. HRM analysis and subsequent sequencing detected pathogenic fully heterozygous APC variants in 10 (6%) of the patients and pathogenic mosaic variants in 2 (1%). All these variants were previously missed by various conventional scanning methods. In parallel, HRM APC scanning was applied to DNA isolated from polyp tissue of two additional patients with apparently sporadic polyposis and without detectable pathogenic APC variant in leukocyte DNA. In both patients a pathogenic mosaic APC variant was present in multiple polyps. The detection of pathogenic APC variants in 7% of the patients, including mosaics, illustrates the usefulness of a complete APC gene reanalysis of previously tested patients, by a supplementary scanning method. HRM is a sensitive and fast pre-screening method for reliable detection of heterozygous and mosaic variants, which can be applied to leukocyte and polyp derived DNA.