To the Editor: Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked multiple congenital anomalies (MCA) and overgrowth syndrome caused by a lossof-function of the glypican-3 gene (GPC3). Almost, a hundred distinct GPC3mutations have been identified, mostly large deletions, nonsense or frameshift mutations. Only 6 intragenic GPC3 duplications were described in SGBS patients, 2 of them detected by array-CGH in patients with MCA. Although their transcriptional effects were assessed in only 2 of them, all these duplications clearly segregated with the SGBS phenotype. Here, we describe the characterization of 2 novel intragenic GPC3 tandem duplications identified by array-CGH during prenatal diagnosis. In both cases increased maternal plasmatic alpha-fetoprotein and abnormal ultrasound findings (polyhydramnios, macrosomia and pyelectasy for the first patient; macrosomia, ventricular septal defect and large hyperechogenic kidneys for the second patient) raised the diagnosis of SGBS. Informed consent for genetic analysis was obtained from the parents according to the French law on Bioethics and following the Helsinki Declaration. In patient 1 (Figure 1A), array-Comparative Genomic Hybridization (array-CGH) revealed a 52-kb Xq26.2 duplication encompassing the exon 7 of GPC3 (arr[hg19] Xq26.2(132 717 085-132 769 148) x2mat). Multiplex ligation-dependent probe analysis (MLPA) confirmed this hemizygous duplication and cDNA sequencing demonstrated a tandem duplication of exon 7. The open reading frame was disrupted by the insertion of 10 amino acids followed by a premature termination codon. In patient 2 (Figure 1A), array-CGH revealed a 825.3-kb Xq26.2 duplication encompassing 3 RefSeq genes (USP26, TFDP3, GPC4) and exons 3 to 8 of GPC3 (arr[hg19] Xq26.2(132 161 510-132 986 815) x2mat). MLPA analysis revealed that only exons 3 to 6 of GPC3 were duplicated while all exons of GPC4 were duplicated. This duplication was composed of 2 adjacent duplications, one involving USP26, TFDP3 and GPC4 while the second involved only the exons 3 to 6 of GPC3. cDNA sequencing conducted after Reverse Transcription-Polymerase Chain Reaction (RT-PCR) showed a tandem orientation of the duplicated exons predicted to cause the addition of 24 amino acids followed by a premature termination codon. In both cases, the diagnosis of SGBS was confirmed postnatally. We reviewed Xq26 duplications registered in public databases (DECIPHER, ISCA and dbVar) (Figure 1B). We excluded the large duplications encompassing the whole GPC3 gene and many other genes in which a loss-of-function of GPC3 is very unlikely. Twelve duplications, partially overlapping GPC3 could potentially disrupt its transcription. Knowing that most interstitial duplications are tandem rearrangements, the 4 duplications totally included within GPC3 (nssv584468, nssv13650346, DECIPHER 258050, DECIPHER 326611) and the 2 duplications with their 30 breakpoint within GPC3 (nssv1415234, nssv13644225) could lead to a GPC3 loss-of-function by disrupting the reading frame. However, features consistent with SGBS were documented in only 2 of them (DECIPHER 258050, nssv13644225) and no functional molecular analysis was performed. The 6 remaining duplications (DECIPHER 253888, nssv1610317, DECIPHER 277238, nssv3397178, nssvs579235 and DECIPHER 278212) either including the whole GPC3 gene or with only a 50 breakpoint in GPC3 probably maintain the production of a full length transcript even if we cannot rule out the presence of a microrearrangement of the gene or a dosage alteration due to a “position effect.” This is in agreement with the clinical data available in the databases which were not suggestive of SGBS. On the other hand, assuming that GPC3 belongs to the class of dosage-sensitive genes, whole GPC3 duplications could give rise to a mirrored SGBS phenotype. However, neither DECIPHER 253888 nor nssv1610317 were documented with clinical features mirroring those of SGBS. In conclusion, this report expands the molecular spectrum of GPC3 mutations in SGBS and illustrates the questions raised in interpreting GPC3 duplications. Array-CGH is useful to determine the exact boundaries of the duplication when one of them involves the 50 or the 30 end of the gene. Nevertheless, as illustrated by our second case, the resolution of array-CGH may be insufficient to detect more complex or intragenic micro-rearrangements within the duplication and a more precise quantitative analysis, such as MLPA, is necessary to determine exactly which genes are involved in the duplication. As microarray analysis is now widely used in both preand postnatal diagnosis, GPC3 duplications might be detected in patients with unexplained developmental problems and/or MCA. If cytogenetics tools such as fluorescence in situ hybridization do not locate the duplicated fragment elsewhere in the genome, or in case of small GPC3 Received: 19 September 2017 Revised: 4 October 2017 Accepted: 5 October 2017

X-linked intellectual disability (XLID) is a clinically and genetically heterogeneous disorder. During the past two decades in excess of 100 X-chromosome ID genes have been identified. Yet, a large number of families mapping to the X-chromosome remained unresolved suggesting that more XLID genes or loci are yet to be identified. Here, we have investigated 405 unresolved families with XLID. We employed massively parallel sequencing of all X-chromosome exons in the index males. The majority of these males were previously tested negative for copy number variations and for mutations in a subset of known XLID genes by Sanger sequencing. In total, 745 X-chromosomal genes were screened. After stringent filtering, a total of 1297 non-recurrent exonic variants remained for prioritization. Co-segregation analysis of potential clinically relevant changes revealed that 80 families (20%) carried pathogenic variants in established XLID genes. In 19 families, we detected likely causative protein truncating and missense variants in 7 novel and validated XLID genes (CLCN4, CNKSR2, FRMPD4, KLHL15, LAS1L, RLIM and USP27X) and potentially deleterious variants in 2 novel candidate XLID genes (CDK16 and TAF1). We show that the CLCN4 and CNKSR2 variants impair protein functions as indicated by electrophysiological studies and altered differentiation of cultured primary neurons from Clcn4−/− mice or after mRNA knock-down. The newly identified and candidate XLID proteins belong to pathways and networks with established roles in cognitive function and intellectual disability in particular. We suggest that systematic sequencing of all X-chromosomal genes in a cohort of patients with genetic evidence for X-chromosome locus involvement may resolve up to 58% of Fragile X-negative cases.

Clinical GeneticsVolume 82, Issue 1 p. 93-96 Aarskog-Scott syndrome: first report of a duplication in the FGD1 gene N Ronce, Corresponding Author N Ronce CHRU de Tours, Service de Génétique, Tours, F-37044, France INSERM U930, Tours, F-37044, France These authors contributed equally to the work.Nathalie RonceCHRU de Tours, Service de Génétique,Tours, F-37044, France INSERM U930,Tours, F-37044, FranceTel: +33 2 47 47 69 35fax: +33 2 47 47 86 53e-mail: [email protected]Search for more papers by this authorI Maystadt, I Maystadt Institut de Pathologie et de Génétique (IPG), Charleroi (Gosselies), B-6041, Belgium Facultés Universitaires Notre-Dame de la Paix (FUNDP), Namur, B-6000, Belgium These authors contributed equally to the work.Search for more papers by this authorC Hubert, C Hubert CHRU de Tours, Service de Génétique, Tours, F-37044, France INSERM U930, Tours, F-37044, FranceSearch for more papers by this authorS Vonwill, S Vonwill CHRU de Tours, Service de Génétique, Tours, F-37044, France INSERM U930, Tours, F-37044, FranceSearch for more papers by this authorK Devriendt, K Devriendt University Hospitals Leuven (KUL), Department of Human Genetics, Leuven, B-3000, BelgiumSearch for more papers by this authorM-P Moizard, M-P Moizard CHRU de Tours, Service de Génétique, Tours, F-37044, France INSERM U930, Tours, F-37044, FranceSearch for more papers by this authorM Raynaud, M Raynaud CHRU de Tours, Service de Génétique, Tours, F-37044, France INSERM U930, Tours, F-37044, FranceSearch for more papers by this author N Ronce, Corresponding Author N Ronce CHRU de Tours, Service de Génétique, Tours, F-37044, France INSERM U930, Tours, F-37044, France These authors contributed equally to the work.Nathalie RonceCHRU de Tours, Service de Génétique,Tours, F-37044, France INSERM U930,Tours, F-37044, FranceTel: +33 2 47 47 69 35fax: +33 2 47 47 86 53e-mail: [email protected]Search for more papers by this authorI Maystadt, I Maystadt Institut de Pathologie et de Génétique (IPG), Charleroi (Gosselies), B-6041, Belgium Facultés Universitaires Notre-Dame de la Paix (FUNDP), Namur, B-6000, Belgium These authors contributed equally to the work.Search for more papers by this authorC Hubert, C Hubert CHRU de Tours, Service de Génétique, Tours, F-37044, France INSERM U930, Tours, F-37044, FranceSearch for more papers by this authorS Vonwill, S Vonwill CHRU de Tours, Service de Génétique, Tours, F-37044, France INSERM U930, Tours, F-37044, FranceSearch for more papers by this authorK Devriendt, K Devriendt University Hospitals Leuven (KUL), Department of Human Genetics, Leuven, B-3000, BelgiumSearch for more papers by this authorM-P Moizard, M-P Moizard CHRU de Tours, Service de Génétique, Tours, F-37044, France INSERM U930, Tours, F-37044, FranceSearch for more papers by this authorM Raynaud, M Raynaud CHRU de Tours, Service de Génétique, Tours, F-37044, France INSERM U930, Tours, F-37044, FranceSearch for more papers by this author First published: 30 December 2011 https://doi.org/10.1111/j.1399-0004.2011.01782.xCitations: 6 Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume82, Issue1July 2012Pages 93-96 RelatedInformation

Moizard M‐P, Ronce N, Blesson S, Bieth E, Burglen L, Mignot C, Mortemousque I, Marmin N, Dessay B, Danesino C, Feillet F, Castelnau P, Toutain A, Moraine C, Raynaud M. Twenty‐five novel mutations including duplications in the ATP7A gene.

X‐linked deafness is a rare cause of hereditary isolated hearing impairment estimated as at least 1% or 2% of the non‐syndromic hearing loss. To date, four loci for DFN have been identified and only one gene, POU3F4 responsible for DFN3, has been cloned. In males, DFN3 is characterized by a progressive deafness associated with perilymphatic gusher at stapes surgery and with a characteristic inner ear malformation. The phenotype of eight independent females carrying POU3F4 anomalies is defined, and a late‐onset hearing loss is found in three patients. Only one has an inner ear malformation. No genotype/phenotype correlation is identified.

A large French family including members affected by nonspecific X-linked mental retardation, with or without autism or pervasive developmental disorder in affected male patients, has been found to have a 2-base-pair deletion in the Neuroligin 4 gene (NLGN4) located at Xp22.33. This mutation leads to a premature stop codon in the middle of the sequence of the normal protein and is thought to suppress the transmembrane domain and sequences important for the dimerization of neuroligins that are required for proper cell-cell interaction through binding to beta-neurexins. As the neuroligins are mostly enriched at excitatory synapses, these results suggest that a defect in synaptogenesis may lead to deficits in cognitive development and communication processes. The fact that the deletion was present in both autistic and nonautistic mentally retarded males suggests that the NLGN4 gene is not only involved in autism, as previously described, but also in mental retardation, indicating that some types of autistic disorder and mental retardation may have common genetic origins.

FG syndrome (OMIM 305450) is an X-linked condition comprising mental retardation, congenital hypotonia, constipation or anal malformations, and a distinctive appearance with disproportionately large head, tall and broad forehead, cowlicks and telecanthus. In a first linkage analysis carried out on 10 families, we demonstrated heterogeneity and assigned one gene [FGS1] to region Xq12-q21.31 [Briault et al., 1997: Am J Med Genet 73:87-90] corroborated by Graham et al. [1998: Am J Med Genet 80:145-156]. Heterogeneity was supported by the study of one family with apparent FG syndrome co-segregating with an inversion of X chromosome [inv(X)(q11q28)] ([FGS2], OMIM 300321) [Briault et al., 1999: Am J Med Genet 86:112-114 and Briault et al., 2000: Am J Med Genet 95:178-181]. We present the results of a new linkage analysis carried out on two families with FG syndrome. The two earlier known loci for FG syndrome, FGS1 and FGS2 (Xq11 or Xq28) were excluded by multipoint analysis of both families. Linkage was found, however, with locus DXS1060 suggesting that a third FG locus might be located at Xp22.3. In this region, two potential candidate genes, VCX-A and PRKX, were excluded by sequence analysis of the coding region in patients of the two reported FG families. The search for new candidate genes is in progress.

The TM4SF2 gene (localized at Xp11.4 between the loci DXS564 and DXS556) has been found to be mutated in one MRX family. In order to define the corresponding behavioral phenotype, global IQ and specific cognitive skills were assessed in seven males and three females of this family, independent of subject status. Mental retardation (MR) was mild in three patients and moderate in three others. Despite the broad variability of severity of MR, a cognitive profile specific to the MR in this family was documented. It was characterized by language disorder that was more marked in the articulatory component and spatial/verbal short-term memory dissociation with larger mnemonic span for spatial than for verbal cues. Linkage analysis was then performed on the basis of the cognitively determined status. Recombinations were observed with the loci DXS556 at Xp11.4 and DXS441 at Xq13.2 (maximum LOD score = 2.23 at theta = 0 for ALAS2). This localization region does not include the TM4SF2 gene that has been found mutated in both patients with MR and in one non-MR male subject of this family. The present results suggest two main hypotheses. First, TM4SF2 gene mutation could be involved in MR in this family, therefore representing accentuated intra familial phenotypic variability. Second, the structural particularity detected in the TM4SF2 gene might reflect a rare polymorphism rather than a pathogenic mutation, with the gene responsible for MR in this family being therefore more likely to be searched for in the pericentromeric region of the X chromosome.

Linkage analysis was performed in three generations of a French family segregating a syndromal form of X-linked mental retardation. All affected males had neonatal hypotonia, seizures, muscular hypodevelopment, and severe mental deficiency. A peak lod score of 2.90 at a recombination fraction of theta = 0 was detected for DXS 1052 and DXS 451 (Xp22.13). Recombination between the disease locus and the polymorphic markers in DXS7163 and DXS1238 suggested a gene mapping to the Xp22.13-Xp21.2 region. Three candidate genes in this region were investigated: the cDNA for kinase Rsk-2 involved in Coffin-Lowry syndrome, the brain-specific exon of a transcript in the DMD locus (DP140 isoform of dystrophin), and exon 18 of the glycerol kinase gene, which is specific to fetal brain transcripts. All three sequences were normal.

Molecular study and neuropsychological analysis were performed concurrently on 49 patients with Duchenne muscular dystrophy (DMD) in order to find a molecular explanation for the cognitive impairment observed in most DMD patients. Complete analysis of the dystrophin gene was performed to define the localization of deletions and duplications in relation to the different DMD promoters. Qualitative analysis of the Dp71 transcript and testing for the specific first exon of Dp140 were also carried out. Neuropsychological analysis assessed verbal and visuospatial intelligence, verbal memory, and reading skills. Comparison of molecular and psychometric findings demonstrated that deletions and duplications that were localized in the distal part of the gene seemed to be preferentially associated with cognitive impairment. Two altered Dp71 transcripts and two deleted Dp140 DNA sequences were found in four patients with severe cerebral dysfunction. These findings suggest that some sequences located in the distal part of the gene and, in particular, some DMD isoforms expressed in the brain may be related to the cognitive impairment associated with DMD.

Linkage analysis was performed in a family with non-specific X-linked mental retardation (MRX 15). Hypotonia in infancy was the most remarkable physical manifestation. The severity of mental deficiency was variable among the patients, but all of them had poor or absent speech. Significant lod scores at a recombination fraction of zero were detected with the marker loci DXS1126, DXS255, and DXS573 (Zmax = 2.01) and recombination was observed with the two flanking loci DXS164 (Xp21.1) and DXS988 (Xp11.22), identifying a 17 cM interval. This result suggests a new gene localization in the proximal Xp region. In numerous families with non-specific X-linked mental retardation (MRX), the corresponding gene has been localized to the paracentromeric region in which a low recombination rate impairs the precision of mapping.

Gene localization was determined by linkage analysis in a large French family with X-linked mental retardation (MRX). Seven living affected males were clinically studied and the clinical picture was characterized by moderate to severe mental handicap with poor secondary speech acquisition. Seizures, slight microcephaly, simian crease, anteverted pinnae, and macroorchidism were observed in some patients only. Linkage analysis revealed no recombination between the MRX gene and two loci: DXS255 at Xp11.22 (Zmax = 3.31 at theta = 0.00) and PGKP1 at Xq11.2-q12 (Zmax = 3.08 at theta = 0.00). One recombination was observed between the gene and the two loci DXS164 at Xp21.2 and DXS441 at Xq13.3, respectively. These results suggested gene localization in the pericentromeric region of the X chromosome, and the LOD scores justified assignment of the symbol MRX14 to this family.