335.4 kb microduplication in chromosome band Xp11.2p11.3 associated with developmental delay, growth retardation, autistic disorder and dysmorphic features

About 10% of causative mutations for mental retardation in male patients involve X chromosome (X-linked mental retardation, XLMR). We describe a case of a 3-year-old boy presenting with developmental delay, autistic features and growth and speech delay. Array-CGH analysis detected a microduplication on the X chromosome (Xp11.2p11.3), spanning 335.4 kb and including 3 known genes (ZNF81, ZNF182 and SPACA5). Genome-wide association studies show that approximately 30% of mutations causing XLMR are located in Xp11.2p11.3, where few pathogenic genes have been identified to date (such as ZNF41, PQB1 and ZNF81). ZNF81 codifies a zinc finger protein and mutations (non-sense mutations, deletions and structural rearrangements) involving this gene have already been described in association with mental retardation. Larger duplications in the same region have also been observed in association with mental retardation, and, in one case, the over-expression of ZNF81 has also been verified by mRNA quantification. No duplications of the single gene have been identified. To our knowledge, the microduplication found in our patient is the smallest ever described in Xp11.2p11.3. This suggests that the over-expression of ZNF81 could have pathological effects.     

SLC9A6 mutations cause X-linked mental retardation, microcephaly, epilepsy, and ataxia, a phenotype mimicking Angelman syndrome

Linkage analysis and DNA sequencing in a family exhibiting an X-linked mental retardation (XLMR) syndrome, characterized by microcephaly, epilepsy, ataxia, and absent speech and resembling Angelman syndrome, identified a deletion in the SLC9A6 gene encoding the Na(+)/H(+) exchanger NHE6. Subsequently, other mutations were found in a male with mental retardation (MR) who had been investigated for Angelman syndrome and in two XLMR families with epilepsy and ataxia, including the family designated as having Christianson syndrome. Therefore, mutations in SLC9A6 cause X-linked mental retardation. Additionally, males with findings suggestive of unexplained Angelman syndrome should be considered as potential candidates for SLC9A6 mutations.     

Monogenic causes of X-linked mental retardation

Mutations in X-linked genes are likely to account for the observation that more males than females are affected by mental retardation. Causative mutations have recently been identified in both syndromic X-linked mental retardation (XLMR) and in the genetically heterogeneous 'nonspecific' forms of XLMR, for which cognitive impairment is the only defining clinical feature. Proteins that function in chromatin remodelling are affected in three important syndromic forms of XLMR. In nonspecific forms of the disorder, defects have been found in signal-transduction pathways that are believed to function during neuronal maturation. These findings provide important insights into the molecular and cellular defects that underlie mental retardation.     

ZNF674: a new kruppel-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.     

X-linked mental retardation: many genes for a complex disorder

X-linked mental retardation (XLMR) is a common cause of moderate to severe intellectual disability in males. XLMR is very heterogeneous, and about two-thirds of patients have clinically indistinguishable non-syndromic (NS-XLMR) forms, which has greatly hampered their molecular elucidation. A few years ago, international consortia overcame this impasse by collecting DNA and cell lines from large cohorts of XLMR families, thereby paving the way for the systematic study of the molecular causes of XLMR. Mutations in known genes might already account for 50% of the families with NS-XLMR, and various genes have been pinpointed that seem to be of particular diagnostic importance. Eventually, even therapy of XLMR might become possible, as suggested by the unexpected plasticity of the neuronal wiring in the brain, and the recent successful drug treatment of a fly model for fragile X syndrome.     

X-linked mental retardation

X-linked mental retardation (XLMR) affects 1.8 per thousand male births and is usually categorized as "syndromic" (MRXS) or "non-specific" (MRX) forms according to the presence or absence of specific signs in addition to the MR. Up to 60 genes have been implicated in XLMR and certain mutations can alternatively lead to MRXS or MRX. Indeed the extreme phenotypic and allelic heterogeneity of XLMR makes the classification of most genes difficult. Therefore, following identification of new genes, accurate retrospective clinical evaluation of patients and their families is necessary to aid the molecular diagnosis and the classification of this heterogeneous group of disorders. Analyses of the protein products corresponding to XLMR genes show a great diversity of cellular pathways involved in MR. Common mechanisms are beginning to emerge : a first group of proteins belongs to the Rho and Rab GTPase signaling pathways involved in neuronal differentiation and synaptic plasticity and a second group is related to the regulation of gene expression. In this review, we illustrate the complexity of XLMR conditions and present recent data about the FMR1, ARX and Oligophrenin 1 genes.     

High prevalence of SLC6A8 deficiency in X-linked mental retardation

A novel X-linked mental retardation (XLMR) syndrome was recently identified, resulting from creatine deficiency in the brain caused by mutations in the creatine transporter gene, SLC6A8. We have studied the prevalence of SLC6A8 mutations in a panel of 290 patients with nonsyndromic XLMR archived by the European XLMR Consortium. The full-length open reading frame and splice sites of the SLC6A8 gene were investigated by DNA sequence analysis. Six pathogenic mutations, of which five were novel, were identified in a total of 288 patients with XLMR, showing a prevalence of at least 2.1% (6/288). The novel pathogenic mutations are a nonsense mutation (p.Y317X) and four missense mutations. Three missense mutations (p.G87R, p.P390L, and p.P554L) were concluded to be pathogenic on the basis of conservation, segregation, chemical properties of the residues involved, as well as the absence of these and any other missense mutation in 276 controls. For the p.C337W mutation, additional material was available to biochemically prove (i.e., by increased urinary creatine : creatinine ratio) pathogenicity. In addition, we found nine novel polymorphisms (IVS1+26G-->A, IVS7+37G-->A, IVS7+87A-->G, IVS7-35G-->A, IVS12-3C-->T, IVS2+88G-->C, IVS9-36G-->A, IVS12-82G-->C, and p.Y498) that were present in the XLMR panel and/or in the control panel. Two missense variants (p.V629I and p.M560V) that were not highly conserved and were not associated with increased creatine : creatinine ratio, one translational silent variant (p.L472), and 10 intervening sequence variants or untranslated region variants (IVS6+9C-->T, IVS7-151_152delGA, IVS7-99C-->A, IVS8-35G-->A, IVS8+28C-->T, IVS10-18C-->T, IVS11+21G-->A, IVS12+15C-->T, *207G-->C, IVS12+32C-->A) were found only in the XLMR panel but should be considered as unclassified variants or as a polymorphism (p.M560V). Our data indicate that the frequency of SLC6A8 mutations in the XLMR population is close to that of CGG expansions in FMR1, the gene responsible for fragile-X syndrome.     

参与X染色体连锁智力障碍的表观遗传因子——PHF8研究进展

PHF8作为JmjC家族中的成员,通过对组蛋白赖氨酸的去甲基化酶活性来调节靶基因的转录。PHF8基因的一系列突变在X染色体连锁智力障碍（XLMR）患者中被发现。主要针对PHF8与XLMR发生的相关性以及PHF8的生化、生理功能进行阐述。     

Mutations in the gene encoding the Sigma 2 subunit of the adaptor protein 1 complex, AP1S2, cause X-linked mental retardation

In a systematic sequencing screen of the coding exons of the X chromosome in 250 families with X-linked mental retardation (XLMR), we identified two nonsense mutations and one consensus splice-site mutation in the AP1S2 gene on Xp22 in three families. Affected individuals in these families showed mild-to-profound mental retardation. Other features included hypotonia early in life and delay in walking. AP1S2 encodes an adaptin protein that constitutes part of the adaptor protein complex found at the cytoplasmic face of coated vesicles located at the Golgi complex. The complex mediates the recruitment of clathrin to the vesicle membrane. Aberrant endocytic processing through disruption of adaptor protein complexes is likely to result from the AP1S2 mutations identified in the three XLMR-affected families, and such defects may plausibly cause abnormal synaptic development and function. AP1S2 is the first reported XLMR gene that encodes a protein directly involved in the assembly of endocytic vesicles.     

Mutations in CUL4B, which encodes a ubiquitin E3 ligase subunit, cause an X-linked mental retardation syndrome associated with aggressive outbursts, seizures, relative macrocephaly, central obesity, hypogonadism, pes cavus, and tremor

We have identified three truncating, two splice-site, and three missense variants at conserved amino acids in the CUL4B gene on Xq24 in 8 of 250 families with X-linked mental retardation (XLMR). During affected subjects' adolescence, a syndrome emerged with delayed puberty, hypogonadism, relative macrocephaly, moderate short stature, central obesity, unprovoked aggressive outbursts, fine intention tremor, pes cavus, and abnormalities of the toes. This syndrome was first described by Cazebas et al., in a family that was included in our study and that carried a CUL4B missense variant. CUL4B is a ubiquitin E3 ligase subunit implicated in the regulation of several biological processes, and CUL4B is the first XLMR gene that encodes an E3 ubiquitin ligase. The relatively high frequency of CUL4B mutations in this series indicates that it is one of the most commonly mutated genes underlying XLMR and suggests that its introduction into clinical diagnostics should be a high priority.     

Mutations in the BRWD3 gene cause X-linked mental retardation associated with macrocephaly

In the course of systematic screening of the X-chromosome coding sequences in 250 families with nonsyndromic X-linked mental retardation (XLMR), two families were identified with truncating mutations in BRWD3, a gene encoding a bromodomain and WD-repeat domain–containing protein. In both families, the mutation segregates with the phenotype in affected males. Affected males have macrocephaly with a prominent forehead, large cupped ears, and mild-to-moderate intellectual disability. No truncating variants were found in 520 control X chromosomes. BRWD3 is therefore a new gene implicated in the etiology of XLMR associated with macrocephaly and may cause disease by altering intracellular signaling pathways affecting cellular proliferation.     

Mutations in ZDHHC9, which encodes a palmitoyltransferase of NRAS and HRAS, cause X-linked mental retardation associated with a Marfanoid habitus

We have identified one frameshift mutation, one splice-site mutation, and two missense mutations in highly conserved residues in ZDHHC9 at Xq26.1 in 4 of 250 families with X-linked mental retardation (XLMR). In three of the families, the mental retardation phenotype is associated with a Marfanoid habitus, although none of the affected individuals meets the Ghent criteria for Marfan syndrome. ZDHHC9 is a palmitoyltransferase that catalyzes the posttranslational modification of NRAS and HRAS. The degree of palmitoylation determines the temporal and spatial location of these proteins in the plasma membrane and Golgi complex. The finding of mutations in ZDHHC9 suggests that alterations in the concentrations and cellular distribution of target proteins are sufficient to cause disease. This is the first XLMR gene to be reported that encodes a posttranslational modification enzyme, palmitoyltransferase. Furthermore, now that the first palmitoyltransferase that causes mental retardation has been identified, defects in other palmitoylation transferases become good candidates for causing other mental retardation syndromes.     

Renpenning syndrome maps to Xp11.

Mutations in genes on the X chromosome are believed to be responsible for the excess of males among individuals with mental retardation. Such genes are numerous, certainly >100, and cause both syndromal and nonsyndromal types of mental retardation. Clinical and molecular studies have been conducted on the Mennonite family with X-linked mental retardation (XLMR) reported, in 1962, by Renpenning et al. The clinical phenotype includes severe mental retardation, microcephaly, up-slanting palpebral fissures, small testes, and stature shorter than that of nonaffected males. Major malformations, neuromuscular abnormalities, and behavioral disturbances were not seen. Longevity is not impaired. Carrier females do not show heterozygote manifestations. The syndrome maps to Xp11.2-p11.4, with a maximum LOD score of 3.21 (recombination fraction 0) for markers between DXS1039 and DXS1068. Renpenning syndrome (also known as "MRXS8"; gene RENS1, MIM 309500) shares phenotypic manifestations with several other XLMR syndromes, notably the Sutherland-Haan syndrome. In none of these entities has the responsible gene been isolated; hence, the possibility that two or more of them may be allelic cannot be excluded at present.     

Clinical genealogical and molecular genetic study of patients with mental retardation

The results of clinical, genealogical, cytogenetic and molecular genetic studies of 113 patients from 96 families with different forms of mental retardation from Ukraine are presented. This study was held as part of the CHERISH project of the 7-th Framework Program. The aim of the project is to improve diagnostics of mental retardation in children in Eastern Europe and Central Asia through detailed analysis of known chromosomal and gene's aberrations and to find the new gene-candidates that cause mental retardation. All patients have normal chromosome number (46XY or 46XX). The cases with fragile-X syndrome were eliminated using molecular genetic methods. Genome rearrangements were found among 28 patients using cytogenetic analysis, multiplex ligation-dependent probe amplification (MLPA analysis) ofsubtelomeric regions and array-based comparative genomic hybridisation (array CGH screening). In 10 cases known pathogenic CNV's were identified, 11 cases are unknown aberrations; their pathogenicity is being determined. The rest cases are known nonpathogenic gene rearrangements. Obtained results show the strong genetic heterogeneity of hereditary forms of mental retardation. The further studies will allow to identificate genes candidates and certain mutations in these genes that may be associated with this pathology.     

Evaluation of point mutations in dystrophin gene in Iranian Duchenne and Becker muscular dystrophy patients: introducing three novel variants

Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked neuromuscular diseases characterized by progressive muscular weakness and degeneration of skeletal muscles. Approximately two-thirds of the patients have large deletions or duplications in the dystrophin gene and the remaining one-third have point mutations. This study was performed to evaluate point mutations in Iranian DMD/BMD male patients. A total of 29 DNA samples from patients who did not show any large deletion/duplication mutations following multiplex polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) screening were sequenced for detection of point mutations in exons 50–79. Also exon 44 was sequenced in one sample in which a false positive deletion was detected by MLPA method. Cycle sequencing revealed four nonsense, one frameshift and two splice site mutations as well as two missense variants.     

Characterisation and Validation of Insertions and Deletions in 173 Patient Exomes

Recent advances in genomics technologies have spurred unprecedented efforts in genome and exome re-sequencing aiming to unravel the genetic component of rare and complex disorders. While in rare disorders this allowed the identification of novel causal genes, the missing heritability paradox in complex diseases remains so far elusive. Despite rapid advances of next-generation sequencing, both the technology and the analysis of the data it produces are in its infancy. At present there is abundant knowledge pertaining to the role of rare single nucleotide variants (SNVs) in rare disorders and of common SNVs in common disorders. Although the 1,000 genome project has clearly highlighted the prevalence of rare variants and more complex variants (e.g. insertions, deletions), their role in disease is as yet far from elucidated.We set out to analyse the properties of sequence variants identified in a comprehensive collection of exome re-sequencing studies performed on samples from patients affected by a broad range of complex and rare diseases (N = 173). Given the known potential for Loss of Function (LoF) variants to be false positive, we performed an extensive validation of the common, rare and private LoF variants identified, which indicated that most of the private and rare variants identified were indeed true, while common novel variants had a significantly higher false positive rate. Our results indicated a strong enrichment of very low-frequency insertion/deletion variants, so far under-investigated, which might be difficult to capture with low coverage and imputation approaches and for which most of study designs would be under-powered. These insertions and deletions might play a significant role in disease genetics, contributing specifically to the underlining rare and private variation predicted to be discovered through next generation sequencing.     

Skewed X-chromosome inactivation is a common feature of X-linked mental retardation disorders

Some deleterious X-linked mutations may result in a growth disadvantage for those cells in which the mutation, when on the active X chromosome, affects cell proliferation or viability. To explore the relationship between skewed X-chromosome inactivation and X-linked mental retardation (XLMR) disorders, we used the androgen receptor X-inactivation assay to determine X-inactivation patterns in 155 female subjects from 24 families segregating 20 distinct XLMR disorders. Among XLMR carriers, ~50% demonstrate markedly skewed X inactivation (i.e., patterns 80:20), compared with only ~10% of female control subjects (P<.001). Thus, skewed X inactivation is a relatively common feature of XLMR disorders. Of the 20 distinct XLMR disorders, 4 demonstrate a strong association with skewed X inactivation, since all carriers of these mutations demonstrate X-inactivation patterns 80:20. The XLMR mutations are present on the preferentially inactive X chromosome in all 20 informative female subjects from these families, indicating that skewing is due to selection against those cells in which the XLMR mutation is on the active X chromosome.     

Automating the identification of DNA variations using quality-based fluorescence re-sequencing: analysis of the human mitochondrial genome.

Diagnostic re-sequencing plays a central role in medical and evolutionary genetics. In this report we describe a process that applies fluorescence-based re-sequencing and an integrated set of analysis tools to automate and simplify the identification of DNA variations using the human mitochondrial genome as a model system. Two programs used in genome sequence analysis (Phred, a base-caller, and Phrap, a sequence assembler) are applied to assess the quality of each base call across the sequence. Potential DNA variants are automatically identified and 'tagged' by comparing the assembled sequence with a reference sequence. We also show that employing the Consed program to display a set of highly annotated reference sequences greatly simplifies data analysis by providing a visual database containing information on the location of the PCR primers, coding and regulatory sequences and previously known DNA variants. Among the 12 genomes sequenced 378 variants including 29 new variants were identified along with two heteroplasmic sites, automatically detected by the PolyPhred program. Overall we document the ease and speed of performing high quality and accurate fluorescence-based re-sequencing on long tracts of DNA as well as the application of new approaches to automatically find and view DNA variants among these sequences.