Dorien Lugtenberg

Duplication of the MECP2 region is a frequent cause of severe mental retardation and progressive neurological symptoms in males.

Loss-of-function mutations of the MECP2 gene at Xq28 are associated with Rett syndrome in females and with syndromic and nonsyndromic forms of mental retardation (MR) in males. By array comparative genomic hybridization (array-CGH), we identified a small duplication at Xq28 in a large family with a severe form of MR associated with progressive spasticity. Screening by real-time quantitation of 17 additional patients with MR who have similar phenotypes revealed three more duplications. The duplications in the four patients vary in size from 0.4 to 0.8 Mb and harbor several genes, which, for each duplication, include the MR-related L1CAM and MECP2 genes. The proximal breakpoints are located within a 250-kb region centromeric of L1CAM, whereas the distal breakpoints are located in a 300-kb interval telomeric of MECP2. The precise size and location of each duplication is different in the four patients. The duplications segregate with the disease in the families, and asymptomatic carrier females show complete skewing of X inactivation. Comparison of the clinical features in these patients and in a previously reported patient enables refinement of the genotype-phenotype correlation and strongly suggests that increased dosage of MECP2 results in the MR phenotype. Our findings demonstrate that, in humans, not only impaired or abolished gene function but also increased MeCP2 dosage causes a distinct phenotype. Moreover, duplication of the MECP2 region occurs frequently in male patients with a severe form of MR, which justifies quantitative screening of MECP2 in this group of patients.

A Post-Hoc Comparison of the Utility of Sanger Sequencing and Exome Sequencing for the Diagnosis of Heterogeneous Diseases

The advent of massive parallel sequencing is rapidly changing the strategies employed for the genetic diagnosis and research of rare diseases that involve a large number of genes. So far it is not clear whether these approaches perform significantly better than conventional single gene testing as requested by clinicians. The current yield of this traditional diagnostic approach depends on a complex of factors that include gene‐specific phenotype traits, and the relative frequency of the involvement of specific genes. To gauge the impact of the paradigm shift that is occurring in molecular diagnostics, we assessed traditional Sanger‐based sequencing (in 2011) and exome sequencing followed by targeted bioinformatics analysis (in 2012) for five different conditions that are highly heterogeneous, and for which our center provides molecular diagnosis. We find that exome sequencing has a much higher diagnostic yield than Sanger sequencing for deafness, blindness, mitochondrial disease, and movement disorders. For microsatellite‐stable colorectal cancer, this was low under both strategies. Even if all genes that could have been ordered by physicians had been tested, the larger number of genes captured by the exome would still have led to a clearly superior diagnostic yield at a fraction of the cost.

Structural variation in Xq28 : MECP2 duplications in 1% of patients with unexplained XLMR and in 2% of male patients with severe encephalopathy

Duplications in Xq28 involving MECP2 have been described in patients with severe mental retardation, infantile hypotonia, progressive spasticity, and recurrent infections. However, it is not yet clear to what extent these and accompanying symptoms may vary. In addition, the frequency of Xq28 duplications including MECP2 has yet to be determined in patients with unexplained X-linked mental retardation and (fe)males with severe encephalopathy. In this study, we used multiplex ligation-dependent probe amplification to screen Xq28 including MECP2 for deletions and duplications in these patient cohorts. In the group of 283 patients with X-linked mental retardation, we identified three Xq28 duplications including MECP2, which suggests that approximately 1% of unexplained X-linked mental retardation may be caused by MECP2 duplications. In addition, we found three additional MECP2 duplications in 134 male patients with mental retardation and severe, mostly progressive, neurological symptoms, indicating that the mutation frequency could be as high as 2% in this group of patients. In 329 female patients, no Xq28 duplications were detected. In total, we assessed 13 male patients with a MECP2 duplication from six unrelated families. Moderate to severe mental retardation and childhood hypotonia was noted in all patients. The majority of the patients also presented with absent speech, seizures, and progressive spasticity as well as ataxia or an ataxic gait and cerebral atrophy, two previously unreported symptoms. We propose to implement DNA copy number testing for MECP2 in the current diagnostic testing in all males with moderate to severe mental retardation accompanied by (progressive) neurological symptoms.

Chromosomal copy number changes in patients with non-syndromic X linked mental retardation detected by array CGH

Several studies have shown that array based comparative genomic hybridisation (CGH) is a powerful tool for the detection of copy number changes in the genome of individuals with a congenital disorder. In this study, 40 patients with non-specific X linked mental retardation were analysed with full coverage, X chromosomal, bacterial artificial chromosome arrays. Copy number changes were validated by multiplex ligation dependent probe amplification as a fast method to detect duplications and deletions in patient and control DNA. This approach has the capacity to detect copy number changes as small as 100 kb. We identified three causative duplications: one family with a 7 Mb duplication in Xp22.2 and two families with a 500 kb duplication in Xq28 encompassing the MECP2 gene. In addition, we detected four regions with copy number changes that were frequently identified in our group of patients and therefore most likely represent genomic polymorphisms. These results confirm the power of array CGH as a diagnostic tool, but also emphasise the necessity to perform proper validation experiments by an independent technique.

Mutations in DYNC1H1 cause severe intellectual disability with neuronal migration defects

Background DYNC1H1 encodes the heavy chain protein of the cytoplasmic dynein 1 motor protein complex that plays a key role in retrograde axonal transport in neurons. Furthermore, it interacts with the LIS1 gene of which haploinsufficiency causes a severe neuronal migration disorder in humans, known as classical lissencephaly or Miller-Dieker syndrome. Aim To describe the clinical spectrum and molecular characteristics of DYNC1H1 mutations. Methods A family based exome sequencing approach was used to identify de novo mutations in patients with severe intellectual disability. Results In this report the identification of two de novo missense mutations in DYNC1H1 (p.Glu1518Lys and p.His3822Pro) in two patients with severe intellectual disability and variable neuronal migration defects is described. Conclusion Since an autosomal dominant mutation in DYNC1H1 was previously identified in a family with the axonal (type 2) form of Charcot- Marie-Tooth (CMT2) disease and mutations in Dync1h1 in mice also cause impaired neuronal migration in addition to neuropathy, these data together suggest that mutations in DYNC1H1 can lead to a broad phenotypic spectrum and confirm the importance of DYNC1H1 in both central and peripheral neuronal functions.

ZNF674: A New Krüppel-Associated Box–Containing Zinc-Finger Gene Involved in Nonsyndromic X-Linked Mental Retardation

Array-based comparative genomic hybridization has proven to be successful in the identification of genetic defects in disorders involving mental retardation. Here, we studied a patient with learning disabilities, retinal dystrophy, and short stature. The family history was suggestive of an X-linked contiguous gene syndrome. Hybridization of full-coverage X-chromosomal bacterial artificial chromosome arrays revealed a deletion of ~1 Mb in Xp11.3, which harbors RP2, SLC9A7, CHST7, and two hypothetical zinc-finger genes, ZNF673 and ZNF674. These genes were analyzed in 28 families with nonsyndromic X-linked mental retardation (XLMR) that show linkage to Xp11.3; the analysis revealed a nonsense mutation, p.E118X, in the coding sequence of ZNF674 in one family. This mutation is predicted to result in a truncated protein containing the Kruppel-associated box domains but lacking the zinc-finger domains, which are crucial for DNA binding. We characterized the complete ZNF674 gene structure and subsequently tested an additional 306 patients with XLMR for mutations by direct sequencing. Two amino acid substitutions, p.T343M and p.P412L, were identified that were not found in unaffected individuals. The proline at position 412 is conserved between species and is predicted by molecular modeling to reduce the DNA-binding properties of ZNF674. The p.T343M transition is probably a polymorphism, because the homologous ZNF674 gene in chimpanzee has a methionine at that position. ZNF674 belongs to a cluster of seven highly related zinc-finger genes in Xp11, two of which (ZNF41 and ZNF81) were implicated previously in XLMR. Identification of ZNF674 as the third XLMR gene in this cluster may indicate a common role for these zinc-finger genes that is crucial to human cognitive functioning.

Arts syndrome is caused by loss-of-function mutations in PRPS1

Arts syndrome is an X-linked disorder characterized by mental retardation, early-onset hypotonia, ataxia, delayed motor development, hearing impairment, and optic atrophy. Linkage analysis in a Dutch family and an Australian family suggested that the candidate gene maps to Xq22.1-q24. Oligonucleotide microarray expression profiling of fibroblasts from two probands of the Dutch family revealed reduced expression levels of the phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1). Subsequent sequencing of PRPS1 led to the identification of two different missense mutations, c.455T-->C (p.L152P) in the Dutch family and c.398A-->C (p.Q133P) in the Australian family. Both mutations result in a loss of phosphoribosyl pyrophosphate synthetase 1 activity, as was shown in silico by molecular modeling and was shown in vitro by phosphoribosyl pyrophosphate synthetase activity assays in erythrocytes and fibroblasts from patients. This is in contrast to the gain-of-function mutations in PRPS1 that were identified previously in PRPS-related gout. The loss-of-function mutations of PRPS1 likely result in impaired purine biosynthesis, which is supported by the undetectable hypoxanthine in urine and the reduced uric acid levels in serum from patients. To replenish low levels of purines, treatment with S-adenosylmethionine theoretically could have therapeutic efficacy, and a clinical trial involving the two affected Australian brothers is currently underway.

Genotype-phenotype correlations in MYCN-related Feingold syndrome.

Feingold syndrome (FS) is the most frequent cause of familial syndromic gastrointestinal atresia and follows autosomal dominant inheritance. FS is caused by germline mutations in or deletions of the MYCN gene. Previously, 12 different heterozygous MYCN mutations and two deletions containing multiple genes including MYCN were described. All these mutations result in haploinsufficiency of both the canonical MYCN protein and the shorter isoform, ΔMYCN. We report 11 novel mutations including seven mutations in exon 2 that result in a premature termination codon (PTC) in the long MYCN transcript. Moreover, we have identified a PTC in exon 1 that only affects the ΔMYCN isoform, without a phenotypic effect. This suggests that mutations in only ΔMYCN do not contribute to the FS. Additionally, we found three novel deletions encompassing MYCN. Together with our previous report we now have a total of four missense mutations in the DNA binding domain, 19 PTCs of which six render the transcript subject to nonsense‐mediated decay (NMD), and five larger deletions in a total of 77 patients. We have reviewed the clinical features of these patients, and found that digital anomalies, e.g., brachymesophalangy and toe syndactyly, are the most consistent features, present in 100% and 97% of the patients, respectively. Small head circumference was present in 89% of the cases. Gastrointestinal atresia remains the most important major congenital anomaly (55%), but cardiac and renal anomalies are also frequent. We suggest that the presence of brachymesophalangy and toe syndactyly in combination with microcephaly is enough to justify MYCN analysis. Hum Mutat 29(9), 1125–1132, 2008.

High resolution profiling of X chromosomal aberrations by array comparative genomic hybridisation.

The causes of mental handicap are highly variable and involve both genetic and environmental factors. Approximately half of the cases have a familial origin, and in 50% of these the genetic defect can be linked to the X chromosome.1 This group of X linked mental retardation patients comprises patients in whom the mental handicap is associated with other clinical features (syndromic or specific mental retardation), and patients in whom the mental deficit is the only consistent clinical or morphogenetic manifestation (non-specific mental retardation, MRX). So far, the molecular basis of MRX is poorly understood because of the extreme genetic heterogeneity of this disorder. Fourteen MRX genes have been identified over the last few years, but each of these account for only a minor fraction of all cases. Since the causative mutation has been identified in only approximately 17% of families,2 it can be estimated that up to 100 MRX genes exist. Most of the currently known MRX genes were identified through the study of microscopically visible X chromosomal abnormalities: OPHN1 , TM4SF2 , ARHGEF6 , and ZNF41 by positional cloning of chromosomal translocation breakpoints,3–6 and FMR2 , IL1RAPL1 , and FACL4 by chromosome deletion mapping.7–9 In addition, disease related genomic deletions have been identified in other MRX genes and in genes associated with numerous other X linked conditions. In our laboratory, four X chromosomal genes associated with a human disorder have been identified based on deletion mapping: REP-1 in choroideremia,10 NDP in Norrie disease,11 POU3F4 in deafness type 3,12 and RPS6KA6 in mental retardation.13 Deletion mapping, however, is a time consuming method, and standard cytogenetic techniques have a limited resolution of approximately 5–10 Mb. It is a common belief that a number of deletions remain below the detection limit of …

Balanced into array: genome-wide array analysis in 54 patients with an apparently balanced de novo chromosome rearrangement and a meta-analysis

High-resolution genome-wide array analysis enables detailed screening for cryptic and submicroscopic imbalances of microscopically balanced de novo rearrangements in patients with developmental delay and/or congenital abnormalities. In this report, we added the results of genome-wide array analysis in 54 patients to data on 117 patients from seven other studies. A chromosome imbalance was detected in 37% of all patients with two-breakpoint rearrangements. In 49% of these patients, the imbalances were located in one or both breakpoint regions. Imbalances were more frequently (90%) found in complex rearrangements, with the majority (81%) having deletions in the breakpoint regions. The size of our own cohort enabled us to relate the presence of an imbalance to the clinical features of the patients by using a scoring system, the De Vries criteria, that indicates the complexity of the phenotype. The median De Vries score was significantly higher (P=0.002) in those patients with an imbalance (5, range 1–9) than in patients with a normal array result (3, range 0–7). This study provides accurate percentages of cryptic imbalances that can be detected by genome-wide array analysis in simple and complex de novo microscopically balanced chromosome rearrangements and confirms that these imbalances are more likely to occur in patients with a complex phenotype.