Eric Rappaport

Identification of ALK as a major familial neuroblastoma predisposition gene

Neuroblastoma is a childhood cancer that can be inherited, but the genetic aetiology is largely unknown. Here we show that germline mutations in the anaplastic lymphoma kinase (ALK) gene explain most hereditary neuroblastomas, and that activating mutations can also be somatically acquired. We first identified a significant linkage signal at chromosome bands 2p23–24 using a whole-genome scan in neuroblastoma pedigrees. Resequencing of regional candidate genes identified three separate germline missense mutations in the tyrosine kinase domain of ALK that segregated with the disease in eight separate families. Resequencing in 194 high-risk neuroblastoma samples showed somatically acquired mutations in the tyrosine kinase domain in 12.4% of samples. Nine of the ten mutations map to critical regions of the kinase domain and were predicted, with high probability, to be oncogenic drivers. Mutations resulted in constitutive phosphorylation, and targeted knockdown of ALK messenger RNA resulted in profound inhibition of growth in all cell lines harbouring mutant or amplified ALK, as well as in two out of six wild-type cell lines for ALK. Our results demonstrate that heritable mutations of ALK are the main cause of familial neuroblastoma, and that germline or acquired activation of this cell-surface kinase is a tractable therapeutic target for this lethal paediatric malignancy.

Common genetic variants on 5p14.1 associate with autism spectrum disorders

Autism spectrum disorders (ASDs) represent a group of childhood neurodevelopmental and neuropsychiatric disorders characterized by deficits in verbal communication, impairment of social interaction, and restricted and repetitive patterns of interests and behaviour. To identify common genetic risk factors underlying ASDs, here we present the results of genome-wide association studies on a cohort of 780 families (3,101 subjects) with affected children, and a second cohort of 1,204 affected subjects and 6,491 control subjects, all of whom were of European ancestry. Six single nucleotide polymorphisms between cadherin 10 (CDH10) and cadherin 9 (CDH9)—two genes encoding neuronal cell-adhesion molecules—revealed strong association signals, with the most significant SNP being rs4307059 (P = 3.4 × 10-8, odds ratio = 1.19). These signals were replicated in two independent cohorts, with combined P values ranging from 7.4 × 10-8 to 2.1 × 10-10. Our results implicate neuronal cell-adhesion molecules in the pathogenesis of ASDs, and represent, to our knowledge, the first demonstration of genome-wide significant association of common variants with susceptibility to ASDs.

Connexin-26 mutations in sporadic and inherited sensorineural deafness

BACKGROUND Hearing impairment affects one infant in 1000 and 4% of people aged younger than 45 years. Congenital deafness is inherited or apparently sporadic. We have shown previously that DFNB1 on chromosome 13 is a major locus for recessive deafness in about 80% of Mediterranean families and that the connexin-26 gene gap junction protein beta2 (GJB2) is mutated in DFNB1 families. We investigated mutations in the GJB2 gene in familial and sporadic cases of deafness. METHODS We obtained DNA samples from 82 families from Italy and Spain with recessive non-syndromic deafness and from 54 unrelated participants with apparently sporadic congenital deafness. We analysed the coding region of the GJB2 gene for mutations. We also tested 280 unrelated people from the general populations of Italy and Spain for the frameshift mutation 35delG. FINDINGS 49% of participants with recessive deafness and 37% of sporadic cases had mutations in the GJB2 gene. The 35delG mutation accounted for 85% of GJB2 mutations, six other mutations accounted for 6% of alleles, and no changes in the coding region of GJB2 were detected in 9% of DFNB1 alleles. The carrier frequency of mutation 35delG among people from the general population was one in 31 (95% CI one in 19 to one in 87). INTERPRETATION Mutations in the GJB2 gene are a major cause of inherited and apparently sporadic congenital deafness. Mutation 35delG is the most common mutation for sensorineural deafness. Identification of 35delG and other mutations in the GJB2 gene should facilitate diagnosis and counselling for the most common genetic form of deafness.

Cornelia de Lange syndrome is caused by mutations in NIPBL, the human homolog of Drosophila melanogaster Nipped-B

Cornelia de Lange syndrome (CdLS; OMIM 122470) is a dominantly inherited multisystem developmental disorder characterized by growth and cognitive retardation; abnormalities of the upper limbs; gastroesophageal dysfunction; cardiac, ophthalmologic and genitourinary anomalies; hirsutism; and characteristic facial features. Genital anomalies, pyloric stenosis, congenital diaphragmatic hernias, cardiac septal defects, hearing loss and autistic and self-injurious tendencies also frequently occur. Prevalence is estimated to be as high as 1 in 10,000 (ref. 4). We carried out genome-wide linkage exclusion analysis in 12 families with CdLS and identified four candidate regions, of which chromosome 5p13.1 gave the highest multipoint lod score of 2.7. This information, together with the previous identification of a child with CdLS with a de novo t(5;13)(p13.1;q12.1) translocation, allowed delineation of a 1.1-Mb critical region on chromosome 5 for the gene mutated in CdLS. We identified mutations in one gene in this region, which we named NIPBL, in four sporadic and two familial cases of CdLS. We characterized the genomic structure of NIPBL and found that it is widely expressed in fetal and adult tissues. The fly homolog of NIPBL, Nipped-B, facilitates enhancer-promoter communication and regulates Notch signaling and other developmental pathways in Drosophila melanogaster.

A genome-wide association study identifies KIAA0350 as a type 1 diabetes gene.

Type 1 diabetes (T1D) in children results from autoimmune destruction of pancreatic beta cells, leading to insufficient production of insulin. A number of genetic determinants of T1D have already been established through candidate gene studies, primarily within the major histocompatibility complex but also within other loci. To identify new genetic factors that increase the risk of T1D, we performed a genome-wide association study in a large paediatric cohort of European descent. In addition to confirming previously identified loci, we found that T1D was significantly associated with variation within a 233-kb linkage disequilibrium block on chromosome 16p13. This region contains KIAA0350, the gene product of which is predicted to be a sugar-binding, C-type lectin. Three common non-coding variants of the gene (rs2903692, rs725613 and rs17673553) in strong linkage disequilibrium reached genome-wide significance for association with T1D. A subsequent transmission disequilibrium test replication study in an independent cohort confirmed the association. These results indicate that KIAA0350 might be involved in the pathogenesis of T1D and demonstrate the utility of the genome-wide association approach in the identification of previously unsuspected genetic determinants of complex traits.

Rare structural variants found in attention-deficit hyperactivity disorder are preferentially associated with neurodevelopmental genes.

Attention-deficit/hyperactivity disorder (ADHD) is a common and highly heritable disorder, but specific genetic factors underlying risk remain elusive. To assess the role of structural variation in ADHD, we identified 222 inherited copy number variations (CNVs) within 335 ADHD patients and their parents that were not detected in 2026 unrelated healthy individuals. Although no excess CNVs, either deletions or duplications, were found in the ADHD cohort relative to controls, the inherited rare CNV-associated gene set was significantly enriched for genes reported as candidates in studies of autism, schizophrenia and Tourette syndrome, including A2BP1, AUTS2, CNTNAP2 and IMMP2L. The ADHD CNV gene set was also significantly enriched for genes known to be important for psychological and neurological functions, including learning, behavior, synaptic transmission and central nervous system development. Four independent deletions were located within the protein tyrosine phosphatase gene, PTPRD, recently implicated as a candidate gene for restless legs syndrome, which frequently presents with ADHD. A deletion within the glutamate receptor gene, GRM5, was found in an affected parent and all three affected offspring whose ADHD phenotypes closely resembled those of the GRM5 null mouse. Together, these results suggest that rare inherited structural variations play an important role in ADHD development and indicate a set of putative candidate genes for further study in the etiology of ADHD.

High-resolution mapping and analysis of copy number variations in the human genome: A data resource for clinical and research applications

We present a database of copy number variations (CNVs) detected in 2026 disease-free individuals, using high-density, SNP-based oligonucleotide microarrays. This large cohort, comprised mainly of Caucasians (65.2%) and African-Americans (34.2%), was analyzed for CNVs in a single study using a uniform array platform and computational process. We have catalogued and characterized 54,462 individual CNVs, 77.8% of which were identified in multiple unrelated individuals. These nonunique CNVs mapped to 3272 distinct regions of genomic variation spanning 5.9% of the genome; 51.5% of these were previously unreported, and >85% are rare. Our annotation and analysis confirmed and extended previously reported correlations between CNVs and several genomic features such as repetitive DNA elements, segmental duplications, and genes. We demonstrate the utility of this data set in distinguishing CNVs with pathologic significance from normal variants. Together, this analysis and annotation provides a useful resource to assist with the assessment of CNVs in the contexts of human variation, disease susceptibility, and clinical molecular diagnostics.

Chromosome 6p22 Locus Associated with Clinically Aggressive Neuroblastoma

BACKGROUND Neuroblastoma is a malignant condition of the developing sympathetic nervous system that most commonly affects young children and is often lethal. Its cause is not known. METHODS We performed a genomewide association study by first genotyping blood DNA samples from 1032 patients with neuroblastoma and 2043 control subjects of European descent using the Illumina HumanHap550 BeadChip. Samples from three independent groups of patients with neuroblastoma (a total of 720 patients) and 2128 control subjects were then genotyped to replicate significant associations. RESULTS We observed a significant association between neuroblastoma and the common minor alleles of three consecutive single-nucleotide polymorphisms (SNPs) at chromosome band 6p22 and containing the predicted genes FLJ22536 and FLJ44180 (P=1.71x10(-9) to 7.01x10(-10); allelic odds ratio, 1.39 to 1.40). Homozygosity for the at-risk G allele of the most significantly associated SNP, rs6939340, resulted in an increased likelihood of the development of neuroblastoma (odds ratio, 1.97; 95% confidence interval, 1.58 to 2.45). Subsequent genotyping of the three 6p22 SNPs in three independent case series confirmed our observation of an association (P=9.33x10(-15) at rs6939340 for joint analysis). Patients with neuroblastoma who were homozygous for the risk alleles at 6p22 were more likely to have metastatic (stage 4) disease (P=0.02), amplification of the MYCN oncogene in the tumor cells (P=0.006), and disease relapse (P=0.01). CONCLUSIONS A common genetic variation at chromosome band 6p22 is associated with susceptibility to neuroblastoma.

Common variations in BARD1 influence susceptibility to high-risk neuroblastoma

We conducted a SNP-based genome-wide association study (GWAS) focused on the high-risk subset of neuroblastoma. As our previous unbiased GWAS showed strong association of common 6p22 SNP alleles with aggressive neuroblastoma, we restricted our analysis here to 397 high-risk cases compared to 2,043 controls. We detected new significant association of six SNPs at 2q35 within the BARD1 locus (Pallelic = 2.35 × 10−9–2.25 × 10−8). We confirmed each SNP association in a second series of 189 high-risk cases and 1,178 controls (Pallelic = 7.90 × 10−7–2.77 × 10−4). We also tested the two most significant SNPs (rs6435862, rs3768716) in two additional independent high-risk neuroblastoma case series, yielding combined allelic odds ratios of 1.68 each (P = 8.65 × 10−18 and 2.74 × 10−16, respectively). We also found significant association with known BARD1 nonsynonymous SNPs. These data show that common variation in BARD1 contributes to the etiology of the aggressive and most clinically relevant subset of human neuroblastoma.

Germline PHOX2B Mutation in Hereditary Neuroblastoma

To the Editor: We read with interest the study by Trochet and colleagues (2004), published in the April 2004 issue of The American Journal of Human Genetics, that described germline mutations of the paired-like homeobox 2B gene (PHOX2B [MIM 603851]) in neuroblastoma (MIM 256700). We have also considered PHOX2B as a candidate gene for predisposition to neuroblastoma, and we now report on a germline PHOX2B mutation in a pedigree with neuroblastoma. However, we also show that there is no evidence for mutation of this gene in eight other pedigrees with neuroblastoma screened to date. We think these data establish PHOX2B as the first bona fide gene that can predispose to neuroblastoma when mutated in the germline, and the findings further emphasize the complex genetics of this important pediatric malignancy. We previously demonstrated linkage of hereditary neuroblastoma to 16p12-13 by use of a genomewide screening strategy (Maris et al. 2002). Positional cloning of a putative 16p12-13 hereditary neuroblastoma-predisposition gene (HNB1) is ongoing, but the critical genomic region for this gene remains large. We had previously considered and excluded other genes known to be mutated in Hirschsprung disease (MIM 142623) and/or in congenital central hypoventilation syndrome (CCHS [MIM 209880]) as candidates for HNB1, because these disorders can occur coincident with both sporadic and hereditary neuroblastoma (Maris et al. 2002). Because of the recent reports that the vast majority of patients with CCHS harbor PHOX2B mutations, including two patients also affected with neuroblastoma (Amiel et al. 2003; Weese-Mayer et al. 2003), we initiated a screen for germline mutations in this gene in our series of pedigrees with neuroblastoma. Oligonucleotide primer pairs flanking the coding regions of exons 1, 2, and 3 of PHOX2B were designed by use of the program Primer 3.0; these primer pairs were used for PCR amplification and bidirectional sequencing of purified PCR products (primer sequences available on request). We screened germline DNA from the proband and an unaffected family member for each of the seven families that showed cosegregation of a 16p haplotype with disease, as well as for two pedigrees that consisted of cousins with neuroblastoma with no cosegregation of 16p marker haplotypes (see Maris et al. [2002] for details of pedigrees). We also sequenced 109 control DNA samples from the Coriell SNP500 Cancer Panel (Coriell Cell Repositories). All sequence aberrations were confirmed by repeat sequencing after cloning of purified PCR products (TOPO TA Cloning Kit [Invitrogen]), and DNA samples from the remaining available members of the pedigree were also screened for the variant. The Children’s Hospital of Philadelphia institutional review board approved this work. A heterozygous single-base deletion (676delG) was discovered in a complex pedigree with neuroblastoma (fig. 1) (see dbSNP Home Page). This family has seven members in three generations affected with neuroblastoma, and two of these individuals were also shown to have Hirschsprung disease. The proband was affected with neuroblastoma, Hirschsprung disease, and neurofibromatosis type 1 (MIM 162200). The putative nonsense mutation 676delG segregated with neuroblastoma through all three generations, and the frameshift was predicted to produce a slightly truncated protein that would no longer code for the second polyalanine tract. This family had previously been shown to cosegregate a 16p12-13 haplotype with neuroblastoma, and the proband was also shown to have an inactivating mutation in NF1 (3775delT) that was not present in either of her parents (Maris et al. 2002). Tumor material was available only for patient 1-001, and the tumor exon 3 sequence remained heterozygous for the 676delG mutation. In addition, loss-of-heterozygosity studies using microsatellite markers (D4S2912, D4S1587, D4S405, D4S2971, and D4S428) that are closely linked to the PHOX2B locus showed no evidence for allelic deletion. The only other sequence variant discovered in the remaining eight pedigrees was a putative SNP (C552T) in pedigree 12 that is not predicted to affect the resultant protein sequence (S184S) (see dbSNP Home Page). This sequence variant was present in the proband but was not detected in the patient’s affected father (no maternal DNA sample was available). It is important to note that neither sequence variant was identified in the bidirectional sequencing of 218 alleles from the control sample set. This strongly suggests that the 676delG sequence variant that segregates with the disease phenotype is a true mutation. The C552T sequence variant, which does not segregate with the disease, is more likely a very rare polymorphism, but we cannot formally exclude the possibility that there might be a functional effect of this presumably neutral polymorphism. Figure 1 Germline PHOX2B mutation in a pedigree segregating neuroblastoma and Hirschsprung disease. A, Family 1 pedigree structure. DNA samples from this family with neuroblastoma were available only for patients with a PHOX2B result shown (wt = wild type; 676delG ... Accumulated data strongly implicate PHOX2B as an essential regulator of normal autonomic nervous system development (Pattyn et al. 1999; Brunet and Pattyn 2002). The discovery of polyalanine-expansion mutations in the majority of patients with CCHS clearly defines a role for this gene in human disease (Amiel et al. 2003; Weese-Mayer et al. 2003), and there appears to be a correlation between the severity of the respiratory symptoms and the length of polyalanine expansion (Weese-Mayer et al. 2003; Matera et al. 2004). Neuroblastoma represents perhaps the most aberrant phenotype that results from abnormal adrenergic tissue development. The rare but well-described synchronous appearance of neuroblastoma with other disorders of the autonomic nervous system has suggested a common genetic etiology often referred to as a “neurocristopathy” (Gaisie et al. 1979; Nemecek et al. 2003). Although other genes implicated in Hirschsprung disease and/or CCHS have not been excluded as hereditary neuroblastoma-predisposition genes (Maris et al. 2002; Perri et al. 2002), our data further establish PHOX2B as an important gene involved in the initiation of neuroblastoma tumorigenesis. However, the fact that the majority of pedigrees studied here do not show PHOX2B mutations clearly implicates locus heterogeneity for hereditary predisposition to neuroblastoma. Assuming that our inferences of linkage to 16p are correct, and in light of the observation of two germline mutations in the proband of the family presented here, we suggest that an oligogenic mechanism for neuroblastoma initiation should be considered, as has been shown for other diseases of neural crest–derived tissues (Gabriel et al. 2002). It is not yet clear if the PHOX2B mutations discovered in patients with hereditary or sporadic neuroblastoma result in gain or loss of protein function. The hypothesis that PHOX2B functions as a tumor suppressor is supported by the potential predicted consequence of the five mutations described, to date, in patients with neuroblastoma. Weese-Mayer and colleagues discovered a nonsense mutation that predicts a significantly truncated protein that would miss most of exon 3, including all of the 20-alanine repeat motif (Weese-Mayer et al. 2003). The frameshift mutation described here is similar to that reported by Amiel and colleagues (2003) in a patient who also had CCHS, Hirschsprung disease, and neuroblastoma, and, in both cases, the changes in reading frame are predicted to abolish the polyalanine tract. Trochet and colleagues (2004) discovered two missense mutations, both of which map to a conserved portion of the homeodomain and thus may interfere with DNA binding. On the other hand, 4p12 is not a known site of frequent allelic deletion in neuroblastoma (Maris and Matthay 1999), and, to date, biallelic inactivation of PHOX2B has not been demonstrated, although far too few cases have been examined to assert this with confidence. Taken together, these data suggest that PHOX2B mutations are involved in the initiation of neuroblastoma tumorigenesis, especially in patients with associated disorders of the autonomic nervous system. Our data also indicate that germline mutational events in this gene are not involved in the majority of hereditary neuroblastoma cases and that alternative genetic events may predispose to tumorigenesis. Examination of additional patients will facilitate the definition of PHOX2B mutation frequency in the genetic and (apparently) sporadic forms of neuroblastoma and will help to clarify the role of PHOX2B mutations in tumor initiation and progression.