A syndromic neurodevelopmental disorder caused by rare variants in PPFIA3.

PPFIA3 encodes the protein-tyrosine phosphatase, receptor-type, F-polypeptide-interacting-protein-alpha-3 (PPFIA3), which is a member of the LAR-protein-tyrosine phosphatase-interacting-protein (liprin) family involved in synapse formation and function, synaptic vesicle transport, and presynaptic active zone assembly. The protein structure and function are evolutionarily well conserved, but human diseases related to PPFIA3 dysfunction are not yet reported in OMIM. Here, we report 20 individuals with rare PPFIA3 variants (19 heterozygous and 1 compound heterozygous) presenting with developmental delay, intellectual disability, hypotonia, dysmorphisms, microcephaly or macrocephaly, autistic features, and epilepsy with reduced penetrance. Seventeen unique PPFIA3 variants were detected in 18 families. To determine the pathogenicity of PPFIA3 variants in vivo, we generated transgenic fruit flies producing either human wild-type (WT) PPFIA3 or five missense variants using GAL4-UAS targeted gene expression systems. In the fly overexpression assays, we found that the PPFIA3 variants in the region encoding the N-terminal coiled-coil domain exhibited stronger phenotypes compared to those affecting the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of fly Liprin-α leads to embryonic lethality. This lethality is partially rescued by the expression of human PPFIA3 WT, suggesting human PPFIA3 function is partially conserved in the fly. However, two of the tested variants failed to rescue the lethality at the larval stage and one variant failed to rescue lethality at the adult stage. Altogether, the human and fruit fly data reveal that the rare PPFIA3 variants are dominant-negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.

Hemizygous variants in protein phosphatase 1 regulatory subunit 3F (PPP1R3F) are associated with a neurodevelopmental disorder characterized by developmental delay, intellectual disability and autistic features.

Protein phosphatase 1 regulatory subunit 3F (PPP1R3F) is a member of the glycogen targeting subunits (GTSs), which belong to the large group of regulatory subunits of protein phosphatase 1 (PP1), a major eukaryotic serine/threonine protein phosphatase that regulates diverse cellular processes. Here, we describe the identification of hemizygous variants in PPP1R3F associated with a novel X-linked recessive neurodevelopmental disorder in 13 unrelated individuals. This disorder is characterized by developmental delay, mild intellectual disability, neurobehavioral issues such as autism spectrum disorder, seizures and other neurological findings including tone, gait and cerebellar abnormalities. PPP1R3F variants segregated with disease in affected hemizygous males that inherited the variants from their heterozygous carrier mothers. We show that PPP1R3F is predominantly expressed in brain astrocytes and localizes to the endoplasmic reticulum in cells. Glycogen content in PPP1R3F knockout astrocytoma cells appears to be more sensitive to fluxes in extracellular glucose levels than in wild-type cells, suggesting that PPP1R3F functions in maintaining steady brain glycogen levels under changing glucose conditions. We performed functional studies on nine of the identified variants and observed defects in PP1 binding, protein stability, subcellular localization and regulation of glycogen metabolism in most of them. Collectively, the genetic and molecular data indicate that deleterious variants in PPP1R3F are associated with a new X-linked disorder of glycogen metabolism, highlighting the critical role of GTSs in neurological development. This research expands our understanding of neurodevelopmental disorders and the role of PP1 in brain development and proper function.

Next-generation phenotyping contributing to the identification of a 4.7 kb deletion in KANSL1 causing Koolen-de Vries syndrome.

Next-generation phenotyping (NGP) is an application of advanced methods of computer vision on medical imaging data such as portrait photos of individuals with rare disorders. NGP on portraits results in gestalt scores that can be used for the selection of appropriate genetic tests, and for the interpretation of the molecular data. Here, we report on an exceptional case of a young girl that was presented at the age of 8 and 15 and enrolled in NGP diagnostics on the latter occasion. The girl had clinical features associated with Koolen-de Vries syndrome (KdVS) and a suggestive facial gestalt. However, chromosomal microarray (CMA), Sanger sequencing, multiplex ligation-dependent probe analysis (MLPA), and trio exome sequencing remained inconclusive. Based on the highly indicative gestalt score for KdVS, the decision was made to perform genome sequencing to also evaluate noncoding variants. This analysis revealed a 4.7 kb de novo deletion partially affecting intron 6 and exon 7 of the KANSL1 gene. This is the smallest reported structural variant to date for this phenotype. The case illustrates how NGP can be integrated into the iterative diagnostic process of test selection and interpretation of sequencing results.

De Novo and Inherited Loss-of-Function Variants in TLK2: Clinical and Genotype-Phenotype Evaluation of a Distinct Neurodevelopmental Disorder.

Next-generation sequencing is a powerful tool for the discovery of genes related to neurodevelopmental disorders (NDDs). Here, we report the identification of a distinct syndrome due to de novo or inherited heterozygous mutations in Tousled-like kinase 2 (TLK2) in 38 unrelated individuals and two affected mothers, using whole-exome and whole-genome sequencing technologies, matchmaker databases, and international collaborations. Affected individuals had a consistent phenotype, characterized by mild-borderline neurodevelopmental delay (86%), behavioral disorders (68%), severe gastro-intestinal problems (63%), and facial dysmorphism including blepharophimosis (82%), telecanthus (74%), prominent nasal bridge (68%), broad nasal tip (66%), thin vermilion of the upper lip (62%), and upslanting palpebral fissures (55%). Analysis of cell lines from three affected individuals showed that mutations act through a loss-of-function mechanism in at least two case subjects. Genotype-phenotype analysis and comparison of computationally modeled faces showed that phenotypes of these and other individuals with loss-of-function variants significantly overlapped with phenotypes of individuals with other variant types (missense and C-terminal truncating). This suggests that haploinsufficiency of TLK2 is the most likely underlying disease mechanism, leading to a consistent neurodevelopmental phenotype. This work illustrates the power of international data sharing, by the identification of 40 individuals from 26 different centers in 7 different countries, allowing the identification, clinical delineation, and genotype-phenotype evaluation of a distinct NDD caused by mutations in TLK2.

New insights into the clinical and molecular spectrum of the novel CYFIP2-related neurodevelopmental disorder and impairment of the WRC-mediated actin dynamics.

PURPOSE: A few de novo missense variants in the cytoplasmic FMRP-interacting protein 2 (CYFIP2) gene have recently been described as a novel cause of severe intellectual disability, seizures, and hypotonia in 18 individuals, with p.Arg87 substitutions in the majority. METHODS: We assembled data from 19 newly identified and all 18 previously published individuals with CYFIP2 variants. By structural modeling and investigation of WAVE-regulatory complex (WRC)-mediated actin polymerization in six patient fibroblast lines we assessed the impact of CYFIP2 variants on the WRC. RESULTS: Sixteen of 19 individuals harbor two previously described and 11 novel (likely) disease-associated missense variants. We report p.Asp724 as second mutational hotspot (4/19 cases). Genotype-phenotype correlation confirms a consistently severe phenotype in p.Arg87 patients but a more variable phenotype in p.Asp724 and other substitutions. Three individuals with milder phenotypes carry putative loss-of-function variants, which remain of unclear pathogenicity. Structural modeling predicted missense variants to disturb interactions within the WRC or impair CYFIP2 stability. Consistent with its role in WRC-mediated actin polymerization we substantiate aberrant regulation of the actin cytoskeleton in patient fibroblasts. CONCLUSION: Our study expands the clinical and molecular spectrum of CYFIP2-related neurodevelopmental disorder and provides evidence for aberrant WRC-mediated actin dynamics as contributing cellular pathomechanism.

Biallelic Mutations in DNAJC12 Cause Hyperphenylalaninemia, Dystonia, and Intellectual Disability.

Phenylketonuria (PKU, phenylalanine hydroxylase deficiency), an inborn error of metabolism, can be detected through newborn screening for hyperphenylalaninemia (HPA). Most individuals with HPA harbor mutations in the gene encoding phenylalanine hydroxylase (PAH), and a small proportion (2%) exhibit tetrahydrobiopterin (BH4) deficiency with additional neurotransmitter (dopamine and serotonin) deficiency. Here we report six individuals from four unrelated families with HPA who exhibited progressive neurodevelopmental delay, dystonia, and a unique profile of neurotransmitter deficiencies without mutations in PAH or BH4 metabolism disorder-related genes. In these six affected individuals, whole-exome sequencing (WES) identified biallelic mutations in DNAJC12, which encodes a heat shock co-chaperone family member that interacts with phenylalanine, tyrosine, and tryptophan hydroxylases catalyzing the BH4-activated conversion of phenylalanine into tyrosine, tyrosine into L-dopa (the precursor of dopamine), and tryptophan into 5-hydroxytryptophan (the precursor of serotonin), respectively. DNAJC12 was undetectable in fibroblasts from the individuals with null mutations. PAH enzyme activity was reduced in the presence of DNAJC12 mutations. Early treatment with BH4 and/or neurotransmitter precursors had dramatic beneficial effects and resulted in the prevention of neurodevelopmental delay in the one individual treated before symptom onset. Thus, DNAJC12 deficiency is a preventable and treatable cause of intellectual disability that should be considered in the early differential diagnosis when screening results are positive for HPA. Sequencing of DNAJC12 may resolve any uncertainty and should be considered in all children with unresolved HPA.

Mutations in EXTL3 Cause Neuro-immuno-skeletal Dysplasia Syndrome.

EXTL3 regulates the biosynthesis of heparan sulfate (HS), important for both skeletal development and hematopoiesis, through the formation of HS proteoglycans (HSPGs). By whole-exome sequencing, we identified homozygous missense mutations c.1382C>T, c.1537C>T, c.1970A>G, and c.2008T>G in EXTL3 in nine affected individuals from five unrelated families. Notably, we found the identical homozygous missense mutation c.1382C>T (p.Pro461Leu) in four affected individuals from two unrelated families. Affected individuals presented with variable skeletal abnormalities and neurodevelopmental defects. Severe combined immunodeficiency (SCID) with a complete absence of T cells was observed in three families. EXTL3 was most abundant in hematopoietic stem cells and early progenitor T cells, which is in line with a SCID phenotype at the level of early T cell development in the thymus. To provide further support for the hypothesis that mutations in EXTL3 cause a neuro-immuno-skeletal dysplasia syndrome, and to gain insight into the pathogenesis of the disorder, we analyzed the localization of EXTL3 in fibroblasts derived from affected individuals and determined glycosaminoglycan concentrations in these cells as well as in urine and blood. We observed abnormal glycosaminoglycan concentrations and increased concentrations of the non-sulfated chondroitin disaccharide D0a0 and the disaccharide D0a4 in serum and urine of all analyzed affected individuals. In summary, we show that biallelic mutations in EXTL3 disturb glycosaminoglycan synthesis and thus lead to a recognizable syndrome characterized by variable expression of skeletal, neurological, and immunological abnormalities.

Nine newly identified individuals refine the phenotype associated with MYT1L mutations.

Both point mutations and deletions of the MYT1L gene as well as microdeletions of chromosome band 2p25.3 including MYT1L are associated with intellectual disability, obesity, and behavioral problems. Thus, MYT1L is assumed to be the-at least mainly-causative gene in the 2p25.3 deletion syndrome. Here, we present comprehensive descriptions of nine novel individuals bearing MYT1L mutations; most of them single nucleotide variants (SNVs). This increases the number of known individuals with causative deletions or SNVs of MYT1L to 51. Since eight of the nine novel patients bear mutations affecting MYT1L only, the total number of such individuals now nearly equals the number of individuals with larger microdeletions affecting additional genes, allowing for a comprehensive phenotypic comparison of these two patient groups. For example, 55% of the individuals with mutations affecting MYT1L only were overweight or obese as compared to 86% of the individuals with larger microdeletions. A similar trend was observed regarding short stature with 5 versus 35%, respectively. However, these differences were nominally significant only after correction for multiple testing, further supporting the hypothesis that MYT1L haploinsufficiency is central to the 2p25.3 deletion phenotype. Most importantly, the large number of individuals with MYT1L mutations presented and reviewed here allowed for the delineation of a more comprehensive clinical picture. Seizures, postnatal short stature, macrocephaly, and microcephaly could be shown to be over-represented among individuals with MYT1L mutations.

Male infant with paternal uniparental diploidy mosaicism and a 46,XX/46,XY karyotype.

A male patient with mosaic paternal uniparental diploidy (PUD) is presented. After birth, the patient presented with hypoglycemia, hemihypertrophy, umbilical hernia, and hepatomegaly. Afterward pancreatic hypertrophy, liver hemangiomas, and cysts were detected sonographically. At the age of 3.5 months, hepatoblastoma was diagnosed. To investigate suspected Beckwith-Wiedemann syndrome (BWS), extensive genetic analyses were performed using DNA from chorionic villus sampling, amniocentesis, and peripheral blood lymphocytes (chromosome analysis, methylation-specific multiplex ligation-dependent probe amplification assays, microsatellite analyses, and single nucleotide polymorphism array analysis). These analyses led to the detection of mosaic PUD. In peripheral blood lymphocytes, a male cell line (46,XY[27]/46,XX[5]) predominated, suggesting a mixture of uniparental isodisomy and heterodisomy. The genetic analyses suggest that the mosaic PUD status was attributable to fertilization of an oocyte by two sperms, with subsequent triploidy rescue giving rise to haploidy, which in turn was rescued. Notably, in the majority of the 28 mosaic PUD patients reported to date, BWS was initially suspected. Mosaic PUD status is associated with a higher risk for a broad range of malignant and benign tumors than in BWS. As tumors can also occur after childhood surveillance into adolescence is indicated. Mosaic PUD must therefore be considered in patients with suspected BWS.

De novo FBXO11 mutations are associated with intellectual disability and behavioural anomalies.

Intellectual disability (ID) has an estimated prevalence of 1.5-2%. In most affected individuals, its genetic basis remains unclear. Whole exome sequencing (WES) studies have identified a multitude of novel causative gene defects and have shown that a large proportion of sporadic ID cases results from de novo mutations. Here, we present two unrelated individuals with similar clinical features and deleterious de novo variants in FBXO11 detected by WES. Individual 1, a 14-year-old boy, has mild ID as well as mild microcephaly, corrected cleft lip and alveolus, hyperkinetic disorder, mild brain atrophy and minor facial dysmorphism. WES detected a heterozygous de novo 1 bp insertion in the splice donor site of exon 3. Individual 2, a 3-year-old boy, showed ID and pre- and postnatal growth retardation, postnatal mild microcephaly, hyperkinetic and restless behaviour, as well as mild dysmorphism. WES detected a heterozygous de novo frameshift mutation. While ten individuals with ID and de novo variants in FBXO11 have been reported as part of larger studies, only one of the reports has some additional clinical data. Interestingly, the latter individual carries the identical mutation as our individual 2 and also displays ID, intrauterine growth retardation, microcephaly, behavioural anomalies, and dysmorphisms. Thus, we confirm deleterious de novo mutations in FBXO11 as a cause of ID and start the delineation of the associated clinical picture which may also comprise postnatal microcephaly or borderline small head size and behavioural anomalies.

Genetic variants in components of the NALCN-UNC80-UNC79 ion channel complex cause a broad clinical phenotype (NALCN channelopathies).

NALCN is a conserved cation channel, which conducts a permanent sodium leak current and regulates resting membrane potential and neuronal excitability. It is part of a large ion channel complex, the "NALCN channelosome", consisting of multiple proteins including UNC80 and UNC79. The predominant neuronal expression pattern and its function suggest an important role in neuronal function and disease. So far, biallelic NALCN and UNC80 variants have been described in a small number of individuals leading to infantile hypotonia, psychomotor retardation, and characteristic facies 1 (IHPRF1, OMIM 615419) and 2 (IHPRF2, OMIM 616801), respectively. Heterozygous de novo NALCN missense variants in the S5/S6 pore-forming segments lead to congenital contractures of the limbs and face, hypotonia, and developmental delay (CLIFAHDD, OMIM 616266) with some clinical overlap. In this study, we present detailed clinical information of 16 novel individuals with biallelic NALCN variants, 1 individual with a heterozygous de novo NALCN missense variant and an interesting clinical phenotype without contractures, and 12 individuals with biallelic UNC80 variants. We report for the first time a missense NALCN variant located in the predicted S6 pore-forming unit inherited in an autosomal-recessive manner leading to mild IHPRF1. We show evidence of clinical variability, especially among IHPRF1-affected individuals, and discuss differences between the IHPRF1- and IHPRF2 phenotypes. In summary, we provide a comprehensive overview of IHPRF1 and IHPRF2 phenotypes based on the largest cohort of individuals reported so far and provide additional insights into the clinical phenotypes of these neurodevelopmental diseases to help improve counseling of affected families.

Chromatin-Remodeling-Factor ARID1B Represses Wnt/ß-Catenin Signaling.

The link of chromatin remodeling to both neurodevelopment and cancer has recently been highlighted by the identification of mutations affecting BAF chromatin-remodeling components, such as ARID1B, in individuals with intellectual disability and cancer. However, the underlying molecular mechanism(s) remains unknown. Here, we show that ARID1B is a repressor of Wnt/ß-catenin signaling. Through whole-transcriptome analysis, we find that in individuals with intellectual disability and ARID1B loss-of-function mutations, Wnt/ß-catenin target genes are upregulated. Using cellular models of low and high Wnt/ß-catenin activity, we demonstrate that knockdown of ARID1B activates Wnt/ß-catenin target genes and Wnt/ß-catenin-dependent transcriptional reporters in a ß-catenin-dependent manner. Reciprocally, forced expression of ARID1B inhibits Wnt/ß-catenin signaling downstream of the ß-catenin destruction complex. Both endogenous and exogenous ARID1B associate with ß-catenin and repress Wnt/ß-catenin-mediated transcription through the BAF core subunit BRG1. Accordingly, mutations in ARID1B leading to partial or complete deletion of its BRG1-binding domain, as is often observed in intellectual disability and cancers, compromise association with ß-catenin, and the resultant ARID1B mutant proteins fail to suppress Wnt/ß-catenin signaling. Finally, knockdown of ARID1B in mouse neuroblastoma cells leads to neurite outgrowth through ß-catenin. The data suggest that aberrations in chromatin-remodeling factors, such as ARID1B, might contribute to neurodevelopmental abnormalities and cancer through deregulation of developmental and oncogenic pathways, such as the Wnt/ß-catenin signaling pathway.

De Novo Mutations in CHAMP1 Cause Intellectual Disability with Severe Speech Impairment.

CHAMP1 encodes a protein with a function in kinetochore-microtubule attachment and in the regulation of chromosome segregation, both of which are known to be important for neurodevelopment. By trio whole-exome sequencing, we have identified de novo deleterious mutations in CHAMP1 in five unrelated individuals affected by intellectual disability with severe speech impairment, motor developmental delay, muscular hypotonia, and similar dysmorphic features including short philtrum and a tented upper and everted lover lip. In addition to two frameshift and one nonsense mutations, we found an identical nonsense mutation, c.1192C>T (p.Arg398*), in two affected individuals. All mutations, if resulting in a stable protein, are predicted to lead to the loss of the functionally important zinc-finger domains in the C terminus of the protein, which regulate CHAMP1 localization to chromosomes and the mitotic spindle, thereby providing a mechanistic understanding for their pathogenicity. We thus establish deleterious de novo mutations in CHAMP1 as a cause of intellectual disability.

Heterozygous HNRNPU variants cause early onset epilepsy and severe intellectual disability.

Pathogenic variants in genes encoding subunits of the spliceosome are the cause of several human diseases, such as neurodegenerative diseases. The RNA splicing process is facilitated by the spliceosome, a large RNA-protein complex consisting of small nuclear ribonucleoproteins (snRNPs), and many other proteins, such as heterogeneous nuclear ribonucleoproteins (hnRNPs). The HNRNPU gene (OMIM *602869) encodes the heterogeneous nuclear ribonucleoprotein U, which plays a crucial role in mammalian development. HNRNPU is expressed in the fetal brain and adult heart, kidney, liver, brain, and cerebellum. Microdeletions in the 1q44 region encompassing HNRNPU have been described in patients with intellectual disability (ID) and other clinical features, such as seizures, corpus callosum abnormalities (CCA), and microcephaly. Recently, pathogenic HNRNPU variants were identified in large ID and epileptic encephalopathy cohorts. In this study, we provide detailed clinical information of five novels and review two of the previously published individuals with (likely) pathogenic de novo variants in the HNRNPU gene including three non-sense and two missense variants, one small intragenic deletion, and one duplication. The phenotype in individuals with variants in HNRNPU is characterized by early onset seizures (6/7), severe ID (6/6), severe speech impairment (6/6), hypotonia (6/7), and central nervous system (CNS) (5/6), cardiac (4/6), and renal abnormalities (3/4). In this study, we broaden the clinical and mutational HNRNPU-associated spectrum, and demonstrate that heterozygous HNRNPU variants cause epilepsy, severe ID with striking speech impairment and variable CNS, cardiac, and renal anomalies.

De novo microdeletions and point mutations affecting SOX2 in three individuals with intellectual disability but without major eye malformations.

Loss-of-function mutations and deletions of the SOX2 gene are known to cause uni- and bilateral anophthalmia and microphthalmia as well as related disorders such as anophthalmia-esophageal-genital syndrome. Thus, anophthalmia/microphthalmia is the primary indication for targeted, "phenotype first" analyses of SOX2. However, SOX2 mutations are also associated with a wide range of non-ocular abnormalities, such as postnatal growth retardation, structural brain anomalies, hypogenitalism, and developmental delay. The present report describes three patients without anophthalmia/microphthalmia and loss-of-function mutations or microdeletions of SOX2 who had been investigated in a "genotype first" manner due to intellectual disability/developmental delay using whole exome sequencing or chromosomal microarray analyses. This result prompted us to perform SOX2 Sanger sequencing in 192 developmental delay/intellectual disability patients without anophthalmia or microphthalmia. No additional SOX2 loss-of-function mutations were detected in this cohort, showing that SOX2 is clearly not a major cause of intellectual disability without anophthalmia/microphthalmia. In our three patients and four further, reported "genotype first" SOX2 microdeletion patients, anophthalmia/microphthalmia was present in less than half of the patients. Thus, SOX2 is another example of a gene whose clinical spectrum is broadened by the generation of "genotype first" findings using hypothesis-free, genome-wide methods. © 2016 Wiley Periodicals, Inc.

Array-based molecular karyotyping in fetal brain malformations: Identification of novel candidate genes and chromosomal regions.

BACKGROUND: For the majority of congenital brain malformations, the underlying cause remains unknown. Recent studies have implicated rare copy number variations (CNVs) in their etiology. METHODS: Here, we used array-based molecular karyotyping to search for causative CNVs in 33 fetuses of terminated pregnancies with prenatally detected brain malformations and additional extracerebral anomalies. RESULTS: In 11 fetuses, we identified 15 CNVs (0.08 Mb to 29.59 Mb), comprising four duplications and eleven deletions. All larger CNVs (> 5 Mb) had also been detected by prenatal conventional karyotyping. None of these CNVs was present in our 1307 healthy in-house controls (frequency < 0.0008). Among these CNVs, we prioritized six chromosomal regions (1q25.1, 5q35.1, 6q25.3-qter, 11p14.3, 15q11.2-q13.1, 18q21.1) due to their previous association with human brain malformations or owing to the presence of a single gene expressed in human brain. Prioritized genes within these regions were UBTD2, SKA1, SVIP, and, most convincingly, GPR52. However, re-sequencing of GPR52 in 100 samples from fetuses with brain malformations or patients with intellectual disability and brain malformations revealed no disease-causing mutation. CONCLUSION: Our study suggests chromosomal regions 1q25.1, 5q35.1, 6q25.3-qter, 11p14.3, 15q11.2-q13.1, and 18q21.1 to be involved in human brain development. Within three of these regions, we suggest UBTD2, GPR52, and SKA1 as possible candidate genes. Because the overall detection rate of array-based molecular karyotyping was slightly higher (23%) than that of conventional prenatal karyotyping (20%), we suggest it's use for prenatal diagnostic testing in fetuses with nonisolated brain malformations.

Genome-wide CNV analysis in 221 unrelated patients and targeted high-throughput sequencing reveal novel causative candidate genes for colorectal adenomatous polyposis.

To uncover novel causative genes in patients with unexplained adenomatous polyposis, a model disease for colorectal cancer, we performed a genome-wide analysis of germline copy number variants (CNV) in a large, well characterized APC and MUTYH mutation negative patient cohort followed by a targeted next generation sequencing (NGS) approach. Genomic DNA from 221 unrelated German patients was genotyped on high-resolution SNP arrays. Putative CNVs were filtered according to stringent criteria, compared with those of 531 population-based German controls, and validated by qPCR. Candidate genes were prioritized using in silico, expression, and segregation analyses, data mining and enrichment analyses of genes and pathways. In 27% of the 221 unrelated patients, a total of 77 protein coding genes displayed rare, nonrecurrent, germline CNVs. The set included 26 candidates with molecular and cellular functions related to tumorigenesis. Targeted high-throughput sequencing found truncating point mutations in 12% (10/77) of the prioritized genes. No clear evidence was found for autosomal recessive subtypes. Six patients had potentially causative mutations in more than one of the 26 genes. Combined with data from recent studies of early-onset colorectal and breast cancer, recurrent potential loss-of-function alterations were detected in CNTN6, FOCAD (KIAA1797), HSPH1, KIF26B, MCM3AP, YBEY and in three genes from the ARHGAP family. In the canonical Wnt pathway oncogene CTNNB1 (ß-catenin), two potential gain-of-function mutations were found. In conclusion, the present study identified a group of rarely affected genes which are likely to predispose to colorectal adenoma formation and confirmed previously published candidates for tumor predisposition as etiologically relevant.