We report an autosomal recessive neurodevelopmental disorder exclusively affecting females carrying biallelic loss-of-function variants in CIZ1, a gene encoding a nuclear matrix protein essential for the maintenance of X-chromosome inactivation. Eight unrelated affected females were identified, whereas male siblings with biallelic variants were asymptomatic. We showed that loss of CIZ1 compromises the maintenance of X-chromosome inactivation, leading to an abnormal overexpression of a subset of X-linked genes. ### Competing Interest Statement S.A. is a co-founder, and X.B. and E.R., are employees of Medigenome. F.A., B.A. and K.B. are employees of Lifera Omics. G.O. and A.R. are employees of Arcensus GmbH. ### Funding Statement This work was carried out within the framework of the FHU GenOMedS with the support of the Health Cooperation Group of University Hospitals of the Great West (GCS HUGO). This work was also supported by the Groupama Foundation. G.S. and G.MN. received funding from the Medical Research Council (MRC) project grant (UKRI577). M.A, R.A., received funding from the King Salman Center for Disability Research through Excellence Research Center (No. KSCDR-ERC-2024-02). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee of CHU de Nantes (Advisory Committee on Information Processing in Research; number CCTIRS: 14.556) gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Known fetal haemoglobin (HbF)-modulating loci explain 10-24% variation of HbF level in Africans with Sickle Cell Disease (SCD), compared to 50% among Europeans. Here, we report fourteen candidate loci from a genome-wide association study (GWAS) of HbF level in patients with SCD from Cameroon, Tanzania, and the United States of America. We present results of cell-based experiments for FLT1 candidate, demonstrating expression in early haematopoiesis and a possible involvement in hypoxia associated HbF induction. Our study employed genotyping arrays that capture a broad range of African and non-African genetic variation and replicated known loci (BCL11A and HBS1L-MYB). We estimated the heritability of HbF level in SCD at 94%, higher than estimated in unselected Europeans, and suggesting a robust capture of HbF-associated loci by these arrays. Our approach, which involved genotype imputation against six reference haplotype panels and association analysis with each of the panels, proved superior over selecting a best-performing panel, evidenced by a substantial proportion of panel-specific (up to 18%) and a low proportion of shared (28%) imputed variants across the panels.

Purpose:To report the retinal phenotype in 2 patients simulating type 2 macular telangiectasis with new variants in CYP2U1 implicated in hereditary spastic paraplegia type 56 (HSP 56). Design:Cross sectional case series study. Participants:Five members of a non-consanguineous family (parents and 3 male children) were investigated. Methods:All family members underwent a full ophthalmic evaluation and multimodal retinal imaging. Two family members demonstrating retinal anomalies underwent additional OCT angiography, dual wavelength autofluorescence and fluorescence lifetime imaging ophthalmoscopy, kinetic perimetry, fundus-correlated microperimetry, electroretinography, and electro-oculography. Whole-exome sequencing was performed in all 5 family members. Main Outcome Measures:To characterize the retinal phenotype in affected patients with variants in CYP2U1, using multimodal imaging: dual-wavelength autofluorescence, fluorescence lifetime, OCT angiography. Results:The 2 siblings with compound heterozygous novel variants c.452C>T; p.(Pro151Leu), c.943C>T; p.(Gln315Ter) in CYP2U1 demonstrated parafoveal loss of retinal transparency and hyperreflectivity to blue light, redistribution of macular pigment to the parafoveal edge, photoreceptor loss, and fluorescence lifetime imaging ophthalmoscopy anomalies: a pattern compatible with that seen in macular telangiectasia type 2 (MacTel). One had manifest neurological abnormalities since early childhood; the second had no neurological abnormalities. Each parent and the third sibling were heterozygous for 1 variant and were neurologically and ophthalmically normal. Conclusions:These CYP2U1 variants are associated with a retinal phenotype very similar to that otherwise specific for MacTel, suggestive of possible links in the etiology and pathogenesis of these diseases. Financial Disclosures:The author(s) have no proprietary or commercial interest in any materials discussed in this article.

The p21-activated kinase (PAK) family of proteins regulates various processes requiring dynamic cytoskeleton organization such as cell adhesion, migration, proliferation, and apoptosis. Among the six members of the protein family, PAK2 is specifically involved in apoptosis, angiogenesis, or the development of endothelial cells. We report a novel de novo heterozygous missense PAK2 variant, p.(Thr406Met), found in a newborn with clinical manifestations of Knobloch syndrome. In vitro experiments indicated that this and another reported variant, p.(Asp425Asn), result in substantially impaired protein kinase activity. Similar findings were described previously for the PAK2 p.(Glu435Lys) variant found in two siblings with proposed Knobloch syndrome type 2 (KNO2). These new variants support the association of PAK2 kinase deficiency with a second, autosomal dominant form of Knobloch syndrome: KNO2.

Enhancers, through the combinatorial action of transcription factors (TFs), dictate both the spatial specificity and the levels of gene expression, and their aberrations can result in diseases. The association between HMX1 and its downstream enhancer with ear deformities has been previously documented. However, the pathogenic variations and molecular mechanisms underlying the bilateral constricted ear (BCE) malformations remain unclear. This study identifies a copy number variation (CNV) encompassing three enhancers that induces BCE. These enhancers, collectively termed the positional identity hierarchical enhancer cluster (PI-HEC), co-regulate spatiotemporal expression of the HMX1 gene in a coordinated and synergistic mode, each displaying unique activity-location-structure characteristics. A thorough exploration of this regulatory locus reveals that specific motif clusters within the PI-HEC variably modulate its activity and specificity, with the high mobility group (HMG) box combined with Coordinator and homeodomain (HD)-TFs notably influencing them respectively. Our findings based on various types of mouse models, reveal that both aberrant Hmx1 expression in the fibroblasts of the basal pinna, originating from neural crest cells, and ectopic expression in the distal pinna structures contribute to the abnormal development of the outer ear, including the cartilage, muscle, and epidermis tissues. This study deepens our understanding of mammalian ear morphogenesis and sheds light on the complexity of gene expression regulation by enhancers and specific sequence motifs.### Competing Interest StatementThe authors have declared no competing interest.

Purpose: 5-methylcytosine RNA modifications are driven by NSUN methyltransferases. Although variants in NSUN2 and NSUN3 were associated with neurodevelopmental diseases, the physiological role of NSUN6 modifications on transfer RNAs and messenger RNAs remained elusive. Methods: We combined exome sequencing of consanguineous families with functional char-acterization to identify a new neurodevelopmental disorder gene. Results: We identified 3 unrelated consanguineous families with deleterious homozygous var-iants in NSUN6. Two of these variants are predicted to be loss-of-function. One maps to the first exon and is predicted to lead to the absence of NSUN6 via nonsense-mediated decay, whereas we showed that the other maps to the last exon and encodes a protein that does not fold correctly. Likewise, we demonstrated that the missense variant identified in the third family has lost its enzymatic activity and is unable to bind the methyl donor S-adenosyl-L-methionine. The affected individuals present with developmental delay, intellectual disability, motor delay, and behavioral anomalies. Homozygous ablation of the NSUN6 ortholog in Drosophila led to locomotion and learning impairment. Conclusion: Our data provide evidence that biallelic pathogenic variants in NSUN6 cause one form of autosomal recessive intellectual disability, establishing another link between RNA modification and cognition. & COPY; 2023 The Authors. Published by Elsevier Inc. on behalf of American College of Medical Genetics and Genomics. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

ObjectiveThis study aims to clinically and genetically assess 30 unrelated consanguineous Pakistani families from various ethnic backgrounds, all exhibiting features of neurodevelopmental disorders (NDDs).MethodsWe conducted clinical, genetic, biochemical, and molecular analyses on 30 consanguineous families with NDDs enrolled from various regions of Pakistan. The likely molecular causes of primary microcephaly and NDDs were identified. Detailed clinical investigations and molecular diagnoses were performed using whole exome sequencing (WES) of the proband, followed by Sanger sequencing for validation and segregation in the available family members of the affected families.ResultsWES identified likely disease-causing homozygous variants in 30 unrelated consanguineous families. Six families presented newly described variants in known NDD-related genes: ABAT (c.1439 T > G; p.Phe480Cys) [OMIM613163], SLC12A6 (c.2865_2865insT; p.Glu955Asnfs*5) [OMIM 218000], SHANK3 (c.1305-3_1,305-2delTT; p.Gln29-_Gly305del) [OMIM 606232], BCKDK (c.356_356insC; p.Gly119Alafs*24) [OMIM 614923], DDHD2 (c.2065G > T; p.Asp689Tyr) [OMIM 615033], ERCC2 (c.1255G > A; p.Glu419Lys) [OMIM 610756]. Additionally, 12 families had previously reported disease-causing variants associated with different types of NDDs: ATRX (c.109C > T; p.Arg37*) [OMIM 309580], GPR56 [ADGRG1] (c.1423C > T; p.Arg475*) [OMIM 606854], NAGLU (c.1694G > A; p.Arg565Gln) [OMIM 252920], DOLK (c.3G > A; p.Met1Ile) [OMIM 610768], GPT2 (c.815C > T; p.Ser272Leu) [OMIM 616281], DYNC1I2 (c.607 + 1G > A; p.?) [OMIM 618492], FBXL3 (c.885delT; p.Leu295Phefs25*) [OMIM 606220], LINGO1 (c.869G > A; p.Arg290His) [OMIM 618103], and ASPM (c.3978G > A; Trp1326*, c.9557C > G; p.Ser3186*, c.6994C > T; p.Arg2332*) [OMIM 608716]. All the identified variants showed segregation compatible with autosomal recessive inheritance.ConclusionIn the present study, we observed a high frequency of ASPM variants in the genetic analysis of 30 consanguineous families exhibiting features of NDDs, particularly those associated with autosomal recessive primary microcephaly. These findings contribute to studies on genotype–phenotype correlation, genetic counseling for families, and a deeper understanding of human brain function and development.

Introduction Hypomyelinating leukodystrophies are a group of genetic disorders, characterised by severe permanent myelin deficiency. Their clinical features include developmental delay with or without neuroregression, nystagmus, central hypotonia, progressing to spasticity and ataxia.HSPD1encodes the HSP60 chaperonin protein, mediating ATP-dependent folding of imported proteins in the mitochondrial matrix. Pathogenic variants inHSPD1have been related to a number of neurological phenotypes, including the dominantly inherited pure hereditary spastic paraplegia (MIM 605280) and the recessively inherited hypomyelinating leukodystrophy 4 (MIM 612233). Subsequently, an additional phenotype of hypomyelinating leukodystrophy has been reported due to de novo heterozygousHSPD1variants. In the current work, we expand the clinical and genetic spectrum of this hypomyelinating disorder by describing a cohort of three patients, being heterozygous forHSPD1variants involving residue Ala536 of HSP60 (the novel p.Ala536Pro variant and the previously reported p.Ala536Val). Methods Clinical and radiological evaluation; whole exome sequencing, in vitro reconstitution assay and patient fibroblast cell lysate analysis. Results Clinical manifestation was of early-onset nystagmus, tremor and hypotonia evolving into spasticity and ataxia and childhood-onset neuroregression in one case. Brain MRI studies revealed diffuse hypomyelination. The 3D protein structure showed these variants to lie in spatial proximity to the previously reported Leu47Val variant, associated with a similar clinical phenotype. In vitro reconstitution assay and patient fibroblast cell lysate analysis demonstrated that these mutants display aberrant chaperonin protein complex assembly. Discussion We provide evidence that impaired oligomerisation of the chaperonin complex might underlie this HSPD1-related phenotype, possibly through exerting a dominant negative effect.

This study aimed to find the molecular basis of Bardet-Biedl syndrome (BBS) in Pakistani consanguineous families. A total of 12 affected families were enrolled. Clinical investigations were performed to access the BBS-associated phenotypes. Whole exome sequencing was conducted on one affected individual from each family. The computational functional analysis predicted the variants’ pathogenic effects and modeled the mutated proteins. Whole-exome sequencing revealed 9 pathogenic variants in six genes associated with BBS in 12 families. The BBS6/MKS was the most common BBS causative gene identified in five families (5/12, 41.6%), with one novel (c.1226G>A, p.Gly409Glu) and two reported variants. c.774G>A, Thr259LeuTer21 was the most frequent BBS6/MMKS allele in three families 3/5 (60%). Two variants, c.223C>T, p.Arg75Ter and a novel, c. 252delA, p.Lys85STer39 were detected in the BBS9 gene. A novel 8bp deletion c.387_394delAAATAAAA, p. Asn130GlyfsTer3 was found in BBS3 gene. Three known variants were detected in the BBS1, BBS2, and BBS7 genes. Identification of novel likely pathogenic variants in three genes reaffirms the allelic and genetic heterogeneity of BBS in Pakistani patients. The clinical differences among patients carrying the same pathogenic variant may be due to other factors influencing the phenotype, including variants in other modifier genes.

Supplementary Figure 6 from Pericentromeric Instability and Spontaneous Emergence of Human Neoacrocentric and Minute Chromosomes in the Alternative Pathway of Telomere Lengthening

PDF file - 2600K, Supplementary Figure S1. Statistical analysis for calling single nucleotide substitutions (SNSs). Supplementary Figure S2. Example of LOH affecting the APC gene in a tubulovillous adenoma (TVA) with moderate dysplasia (polyp MS1). Supplementary Figure S3. LOH and copy number alterations (CNAs) for all polyps. The data are shown using circos plots. Supplementary Figure S4. Amplification of the MYC locus in an adenoma with severe dysplasia. Supplementary Figure S5. Calculation of mutation rates in normal colon tissue and adenomas. Supplementary Table S1. Clinical, histopathological and sequence coverage data for the examined polyps. Supplementary Table S2. Statistical analysis of cancer-driver genes. Supplementary Table S4. Coding lengths of 19,075 protein-coding genes distributed in 8 bins according to gene size. Supplementary Table S5. Genomic lengths of 19,075 protein-coding genes distributed in 8 bins according to gene size

Telomere maintenance 2 (TELO2), Tel2 interacting protein 2 (TTI2), and Tel2 interacting protein 1 (TTI1) are the three components of the conserved Triple T (TTT) complex that modulates activity of phosphatidylinositol 3-kinase-related protein kinases (PIKKs), including mTOR, ATM, and ATR, by regulating the assembly of mTOR complex 1 (mTORC1). The TTT complex is essential for the expression, maturation, and stability of ATM and ATR in response to DNA damage. TELO2- and TTI2-related bi-allelic autosomal-recessive (AR) encephalopathies have been described in individuals with moderate to severe intellectual disability (ID), short stature, postnatal microcephaly, and a movement disorder (in the case of variants within TELO2). We present clinical, genomic, and functional data from 11 individuals in 9 unrelated families with bi-allelic variants in TTI1. All present with ID, and most with microcephaly, short stature, and a movement disorder. Functional studies performed in HEK293T cell lines and fibroblasts and lymphoblastoid cells derived from 4 unrelated individuals showed impairment of the TTT complex and of mTOR pathway activity which is improved by treatment with Rapamycin. Our data delineate a TTI1-related neurodevelopmental disorder and expand the group of disorders related to the TTT complex.

Supplementary Figure 5 from Pericentromeric Instability and Spontaneous Emergence of Human Neoacrocentric and Minute Chromosomes in the Alternative Pathway of Telomere Lengthening

Supplementary Methods, Legends and Table 1 from Pericentromeric Instability and Spontaneous Emergence of Human Neoacrocentric and Minute Chromosomes in the Alternative Pathway of Telomere Lengthening