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PURPOSE: WNK3 kinase (PRKWNK3) has been implicated in the development and function of the brain via its regulation of the cation-chloride cotransporters, but the role of WNK3 in human development is unknown. METHOD: We ascertained exome or genome sequences of individuals with rare familial or sporadic forms of intellectual disability (ID). RESULTS: We identified a total of 6 different maternally-inherited, hemizygous, 3 loss-of-function or 3 pathogenic missense variants (p.Pro204Arg, p.Leu300Ser, p.Glu607Val) in WNK3 in 14 male individuals from 6 unrelated families. Affected individuals had identifier with variable presence of epilepsy and structural brain defects. WNK3 variants cosegregated with the disease in 3 different families with multiple affected individuals. This included 1 large family previously diagnosed with X-linked Prieto syndrome. WNK3 pathogenic missense variants localize to the catalytic domain and impede the inhibitory phosphorylation of the neuronal-specific chloride cotransporter KCC2 at threonine 1007, a site critically regulated during the development of synaptic inhibition. CONCLUSION: Pathogenic WNK3 variants cause a rare form of human X-linked identifier with variable epilepsy and structural brain abnormalities and implicate impaired phospho-regulation of KCC2 as a pathogenic mechanism.

X-linked creatine transporter deficiency is caused by the deficiency of the creatine transporter encoded by theSLC6A8gene on Xq28. We here report a 3-year-old boy with global developmental delay, autism and epilepsy. He had a normal MRI of the brain. Brain magnetic resonance spectroscopy (MRS) subsequently showed an abnormally small creatine peak. His high urine creatine/creatinine ratio further suggested the diagnosis, later confirmed by hemizygous mutation detected in theSLC6A8gene. His mother was also heterozygous for the same mutation. Supplementation with creatine monohydrate, arginine, and glycine (precursors of creatine) and supportive therapies, resulted in modest clinical improvement after 12 months. This case highlights the importance of doing MRS for boys with global delay/intellectual disability, autism and epilepsy even with a normal MRI of the brain, to pick up a potentially treatable cause.

BackgroundHypohidrotic Ectodermal Dysplasia (HED) is a genetic disorder which affects structures of ectodermal origin. X-linked hypohidrotic ectodermal dysplasia (XLHED) is the most common form of disease. XLHED is characterized by hypotrichosis, hypohydrosis and hypodontia. The cardinal features of classic HED become obvious during childhood.Identification of a hemizygous EDA pathogenic variant in an affected male confirms the diagnosis.Case presentationWe report on a male newborn with the main clinical characteristics of the X-linked HED including hypotrichosis, hypodontia and hypohidrosis. Gene panel sequencing identified a new hemizygous missense variant of uncertain significance (VUS) c.1142G > C (p.Gly381Ala) in the EDA gene, located on the X chromosome and inherited from the healthy mother.ConclusionDespite the potential functional impact of VUS remains uncharacterized, our goal is to evaluate the clinical potential consequences of missense VUS on EDA gene. Even if the proband’s phenotype is characteristic for classic HED, further reports of patients with same clinical phenotype and the same genomic variant are needed to consider this novel VUS as responsible for the development of HED.

We report a 5-year-old male with a PDHA1 variant who presented with alternating hemiplegia of childhood and later developed developmental regression, basal ganglia injury and episodic lactic acidosis. Enzyme assay in lymphocytes confirmed a diagnosis of Pyruvate Dehydrogenase Complex (PDC) deficiency. His mother who was heterozygous for the same variant suffered from ophthalmoplegia, chronic migraine and developed flaccid paralysis at 36 years of age. PDHA1 is the most common genetic cause of PDC deficiency and presents with a myriad of neurological phenotypes including neonatal form with lactic acidosis, non-progressive infantile encephalopathy, Leigh syndrome subtype and intermittent ataxia. The presentations in our 2 patients contribute to the clinical heterogeneity of this neurogenetic condition.

Asthenoteratozoospermia characterized by multiple morphological abnormalities of the flagella (MMAF) has been identified as a sub-type of male infertility. Recent progress has identified several MMAF-associated genes with an autosomal recessive inheritance in human affected individuals, but the etiology in approximately 40% of affected individuals remains unknown. Here, we conducted whole-exome sequencing (WES) and identified hemizygous missense variants in the X-linked CFAP47 in three unrelated Chinese individuals with MMAF. These three CFAP47 variants were absent in human control population genome databases and were predicted to be deleterious by multiple bioinformatic tools. CFAP47 encodes a cilia- and flagella-associated protein that is highly expressed in testis. Immunoblotting and immunofluorescence assays revealed obviously reduced levels of CFAP47 in spermatozoa from all three men harboring deleterious missense variants of CFAP47. Furthermore, WES data from an additional cohort of severe asthenoteratozoospermic men originating from Australia permitted the identification of a hemizygous Xp21.1 deletion removing the entire CFAP47 gene. All men harboring hemizygous CFAP47 variants displayed typical MMAF phenotypes. We also generated a Cfap47-mutated mouse model, the adult males of which were sterile and presented with reduced sperm motility and abnormal flagellar morphology and movement. However, fertility could be rescued by the use of intra-cytoplasmic sperm injections (ICSIs). Altogether, our experimental observations in humans and mice demonstrate that hemizygous mutations in CFAP47 can induce X-linked MMAF and asthenoteratozoospermia, for which good ICSI prognosis is suggested. These findings will provide important guidance for genetic counseling and assisted reproduction treatments.

Kabuki syndrome (KS) is a rare genetic disorder characterized by the following 5 crucial symptoms: dysmorphic facial features, growth retardation, skeletal abnormalities, intellectual disability, and dermatoglyphic malformations. Studies show that most of the KS cases are caused by mutations or large deletions in the KMT2D gene, while the other cases show mutations in KDM6A. We studied 2 patients with suspected KS in 2 unrelated families by whole-exome sequencing to identify the possible genetic cause(s) and by Sanger sequencing to validate the identified variants and check the segregation in other members of the families. Finally, the potential effects of the variants on the structure and function of respective proteins were tested using in silico predictions. Both affected members of the families showed typical manifestations of KS including intellectual disability, developmental delay, and abnormal facial characteristics. A novel heterozygous frameshift variant in the KMT2D gene, c.4981del; p.(Glu1661Serfs*61), and a novel hemizygote missense variant in the KDM6A gene, c.3301G>A; p.(Glu1101Lys), were detected in patients 1 and 2, respectively. The frameshift variant identified in the first family was de novo, while in the second family, the mother was also heterozygous for the missense variant. The frameshift variant in KMT2D is predicted to lead to a truncated protein which is functionally impaired. The Glu1101 residue of KDM6A (UTX) affected in the second patient is located in a conserved region on the surface of the Jumonji domain and predicted to be causative. Our findings provide evidence on the possible pathogenicity of these 2 variants; however, additional functional studies are necessary to confirm their impacts.

Interpretation of the significance of maternally inherited X chromosome variants in males with neurocognitive phenotypes continues to present a challenge to clinical geneticists and diagnostic laboratories. Here we report 14 males from 9 families with duplications at the Xq13.2-q13.3 locus with a common facial phenotype, intellectual disability (ID), distinctive behavioral features, and a seizure disorder in two cases. All tested carrier mothers had normal intelligence. The duplication arose de novo in three mothers where grandparental testing was possible. In one family the duplication segregated with ID across three generations. RLIM is the only gene common to our duplications. However, flanking genes duplicated in some but not all the affected individuals included the brain-expressed genes NEXMIF, SLC16A2, and the long non-coding RNA gene FTX. The contribution of the RLIM-flanking genes to the phenotypes of individuals with different size duplications has not been fully resolved. Missense variants in RLIM have recently been identified to cause X-linked ID in males, with heterozygous females typically having normal intelligence and highly skewed X chromosome inactivation. We detected consistent and significant increase of RLIM mRNA and protein levels in cells derived from seven affected males from five families with the duplication. Subsequent analysis of MDM2, one of the targets of the RLIM E3 ligase activity, showed consistent downregulation in cells from the affected males. All the carrier mothers displayed normal RLIM mRNA levels and had highly skewed X chromosome inactivation. We propose that duplications at Xq13.2-13.3 including RLIM cause a recognizable but mild neurocognitive phenotype in hemizygous males.

s Background Duchenne muscular dystrophies (DMDs) are X-linked recessive neuromuscular disorders with malfunction or absence of the Dystrophin protein. Precise genetic diagnosis is critical for proper planning of patient care and treatment. In this study, we described a Chinese family with mosaic DMD mutations and discussed the best method for prenatal diagnosis and genetic counseling of X-linked familial disorders. Methods We investigated all variants of the whole dystrophin gene using multiple DNA samples isolated from the affected family and identified two variants of the DMD gene in a sick boy and two female carriers by targeted next generation sequencing (TNGS), Sanger sequencing, and haplotype analysis. Results We identified the hemizygous mutation c.6794delG (p.G2265Efs*6) of DMD in the sick boy, which was inherited from his mother. Unexpectedly, a novel heterozygous mutation c.6796delA (p.I2266Ffs*5) of the same gene, which was considered to be a de novo variant, was detected from his younger sister instead of his mother by Sanger sequencing. However, further NGS analysis of the mother and her amniotic fluid samples revealed that the mother carried a low-level mosaic c.6796delA mutation. Conclusions We reported two different mutations of the DMD gene in two siblings, including the novel mutation c.6796delA (p.I2266Ffs*5) inherited from the asymptomatic mosaic-carrier mother. This finding has enriched the knowledge of the pathogenesis of DMD. If no mutation is detected in obligate carriers, the administration of intricate STR/NGS/Sanger analysis will provide new ideas on the prenatal diagnosis of DMD.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common hereditary disorder in China. The existing prevalence and molecular epidemiology of G6PD deficiency in China were geographically limited. In this study, the spectrum of G6PD gene mutations was well characterized in a large and diverse population all over the country; and the correlation of genotype and enzyme activity phenotype was explored for the first time. The results showed that the overall prevalence of G6PD deficiency in China was 2.10% at the national level. The top six common mutations were c.1388 G>A, c.1376 G>T, c.95 A>G, c.392 G>T, c.871 G>A and c.1024 C>T, accounting for more than 90% of G6PD deficient alleles. Compound mutation patterns were frequently observed in females with severe deficiency. The distribution of G6PD activities depended on the type of mutation patterns and genders. Hemizygote, homozygote, and compound heterozygote were predominantly associated with severe G6PD deficiency, whereas heterozygotes with single mutation mainly presented moderate enzyme deficiency. A significant gap between G6PD activities in hemizygous and normal males was observed, and yet, the overall distribution of that in females carrying missense mutations was a continuum from G6PD severely deficient to normal. This is the first report of discussing the association between G6PD genetic variants in the Chinese and enzyme activity phenotypes.

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, are the major cause of X-linked retinitis pigmentosa (RP), in which exon open reading frame 15 (ORF15) of RPGR has been implicated to play a substantial role. We identified a novel hemizygous missense mutation E585K of RPGR from whole-exome sequencing of RP. RNA-Seq analysis and functional study were conducted to investigate the underlying pathogenic mechanism of the mutation. Our results showed that the mutation actually affected RPGR ORF15 splicing. RNA-Seq analysis of the human retina followed by validation in cells revealed a complex splicing pattern near the 3′ boundary of RPGR exon 14 in the ORF15 region, resulting from a variety of alternative splicing events (ASEs). The wildtype RPGR mini-gene expressed in human 293T cells confirmed these ASEs in vitro. In contrast, without new RNA species detected, the mutant mini-gene disrupted the splicing pattern of the ORF15 region, and caused loss of RPGR transcript heterogeneity. The RNA species derived from the mutant mini-gene were predominated by a minor out-of-frame transcript that was also observed in wildtype RPGR, resulting from an upstream alternative 5′ splice site in exon 14. Our findings therefore provide insights into the influence of RPGR exonic mutations on alternative splicing of the ORF15 region, and the underlying molecular mechanism of RP.

The androgen insensitivity syndrome (AIS) is a congenital disease characterized by androgen resistance due to androgen receptor (AR) gene mutations, resulting in disorders of sex differentiation in 46,XY individuals. However, the underlying mechanisms in the majority of AR variants and the phenotype-genotype correlations are unclear. Here, we identified a p.Y764H variant of the AR gene that results in different phenotypes in a family. Structural analyses revealed that amino acid substitution affected protein properties and spatial conformation, and in vitro, functional studies showed impaired nuclear translocation ability of the mutated protein. Moreover, the extent to which this variant reduced nuclear translocation depends on the dihydrotestosterone (DHT) concentrations. Our results, for the first time, demonstrated a pathogenesis of the p.Y764H mutations in AR resulting in AIS phenotype, and indicated that AIS patients with p.Y764H mutation and preserved gonad might have residual AR activity at high androgen levels, putting patients at risk for pubertal virilization in the future. We provide an in-depth insight into the pathogenesis in AIS based on the amino acid substitution, which may help aid its precise diagnosis, personalized treatment, and organized follow-up to avoid gender dysphoria.

Background Female Dent disease 1 patients with low-molecular-weight proteinuria (LMWP) due to CLCN5 gene mutation were rarely reported, and these cases that the people were also with Turner syndrome (TS) were even hardly documented before. Case presentation Here we report a 3-year and 11-month old Chinese girl with short stature who had a karyotype of 46,X,i(X)(q10) and a de novo pathogenic variant in the CLCN5 gene on the short arm of X chromosome. Laboratory examinations showed that the patient had LMWP, hypercalciuria, hypophosphatemia, delayed bone age, and genital dysplasia. Conclusion The combination of i(X)(q10) and CLCN5 mutation causes the deletion of the wild-type CLCN5 allele that results in Dent-1 and TS. To the best of our knowledge, this is the first case that a female CLCN5 mutation hemizygote is diagnosed with Dent-1 and Turner syndrome due to isochromosome X. Also, our case has indicated that the prevalence of the situation may be largely underestimated because of the mild signs of females with Dent-1.

Tafenoquine has been licensed for the single-dose radical cure of Plasmodium vivax in adults; however, it is only recommended in patients with > 70% of normal glucose-6-phosphate dehydrogenase (G6PD) activity. Because this may hinder widespread use, we investigated sex-based treatment strategies in which all adult patients are tested with a qualitative G6PD rapid diagnostic test (RDT). Glucose-6-phosphate dehydrogenase normal males are prescribed tafenoquine in all three strategies, whereas G6PD normal females are prescribed either a low-dose 14-day primaquine regimen (PQ14, total dose 3.5 mg/kg) or a high-dose 7-day primaquine regimen (PQ7, total dose 7 mg/kg), or referred to a healthcare facility for quantitative G6PD testing before prescribing tafenoquine. Patients testing G6PD deficient are prescribed a weekly course of primaquine for 8 weeks. We compared the cost-effectiveness of these three strategies to usual care in four countries using a decision tree model. Usual care in Ethiopia does not include radical cure, whereas Afghanistan, Indonesia, and Vietnam prescribe PQ14 without G6PD screening. The cost per disability-adjusted life-year (DALY) averted was expressed through incremental cost-effectiveness ratios (ICERs). Compared with usual care, the ICERs for a sex-based treatment strategy with PQ7 for females from a healthcare provider perspective were $127 per DALY averted in Vietnam, $466 in Ethiopia, $1,089 in Afghanistan, and $4,443 in Indonesia. The PQ14 and referral options cost more while averting fewer DALYs than PQ7. This study provides an alternative cost-effective mode of rolling out tafenoquine in areas where initial testing with only a G6PD RDT is feasible.

Hemophilia B is an X-linked recessive bleeding disorder caused by diverse mutations throughout the F9 gene. The same F9 mutation may result in different degrees of clotting factor deficiency. The aim of this study was to investigate the pathogenesis of two hemophilia B patients with different severity in a family. A family with two hemophilia B patients was recruited in this study. Coagulation assays, activities of FVIII (FVIII:C) and FIX (FIX:C) were evaluated. All of the exons and intron exon boundaries of the F9 gene were amplified by PCR and analyzed by direct sequencing. The proband, 12-year-old boy with moderate bleeding history, had manifest prolonged activated partial thromboplastin time (98.1 s) and markedly decreased FIX activity (1%). His maternal uncle presented slightly prolonged activated partial thromboplastin time (48.2 s) and mildly decreased FIX activity (15.2%). Molecular genetic analysis of F9 revealed that they were hemizygous for a novel missense mutation, c.157G>C (p.Glu53Gln). Our study widens the mutation spectrum of the FIX gene. In addition, this report provides a specific case associated with genotype and phenotype heterogeneity of hemophilia B.

The aim of this study was to assess the performance of fluorescence in situ hybridization (FISH) in identifying the copy number profiles of the three key peritoneal mesothelioma tumor suppressor genes BAP1, CDKN2A, and NF2, with particular emphasis on minute homozygous deletions, a copy number abnormality recently unveiled at the 3p21 (BAP1) chromosomal region using high-throughput methods. FISH was performed on 75 formalin-fixed-paraffin-embedded peritoneal mesotheliomas and recognized two types of monoallelic loss (monosomy, and hemizygous deletion) and two types of biallelic loss (canonical homozygous deletion with a complete loss of FISH signal and homozygous deletion with diminished signal). Diminished FISH signals revealed deletions occurring within the genomic region covered by the gene-specific probe and affected all three tumor suppressors. BAP1 homozygous deletions with diminished signal outnumbered canonical homozygous deletions (13 vs 3): conversely, canonical homozygous deletions were prevalent for CDKN2A (2 vs 14). Diminished signal homozygous deletion was the only pattern of biallelic loss observed for NF2 (2 cases). Hemizygous deletion mainly affected BAP1 (21 vs 6), while monosomy was prevalent for CDKN2A (14 vs 7) and particularly for NF2 where it accounts for all monoallelic losses. FISH/immunohistochemistry (BAP1, CDKN2A, and MTAP) correlation showed that all homozygous deletions, including those with diminished signals, resulted in a null BAP1 and CDKN2A immunophenotype but only canonical CDKN2A homozygous deletions resulted in MTAP loss of expression. BAP1 hemizygous deletion, but not monosomy, was also invariably associated with loss of protein expression whereas neither type of CDKN2A monoallelic loss correlated with p16 or MTAP immunohistochemistry. Array comparative genomic hybridization performed on a spontaneously emerging peritoneal mesothelioma cell line provided support for the interpretation of the FISH patterns and allowed us to extend the number of chromatin remodeling factors involved in mesothelioma to SETD7 and PCGF5, two previously unreported genes.

Neurofibromatosis type 2 (NF2) gene, a tumor suppressor gene located on chromosome 22q12.2, is frequently abnormal in mesothelioma. Recent studies have revealed the effectiveness of diagnostic assays for differentiating malignant pleural mesothelioma from reactive mesothelial hyperplasia. These include detection of homozygous deletion of the 9p21 locus by fluorescence in situ hybridization (FISH) (9p21 FISH), loss of expression of BAP1 as detected by immunohistochemistry, and loss of expression of methylthioadenosine phosphorylase (MTAP) as detected by immunohistochemistry. However, the application of FISH detection of NF2 gene deletion (NF2 FISH) in differentiation of malignant pleural mesothelioma from reactive mesothelial hyperplasia has not been fully evaluated. In this study, we investigated whether NF2 FISH, either alone or in a combination with other diagnostic assays (9p21 FISH, MTAP immunohistochemistry, and BAP1 immunohistochemistry), is effective for distinguishing malignant pleural mesothelioma from reactive mesothelial hyperplasia. This study cohort included malignant pleural mesothelioma (n = 47) and reactive mesothelial hyperplasia cases (n = 27) from a period between 2001 and 2017. We used FISH to examine deletion status of NF2 and 9p21 and immunohistochemistry to examine expression of MTAP and BAP1 in malignant pleural mesothelioma and in reactive mesothelial hyperplasia. Hemizygous NF2 loss (chromosome 22 monosomy or hemizygous deletion) was detected in 25 of 47 (53.2%) mesothelioma cases. None of the mesothelioma cases showed homozygous NF2 deletion. Hemizygous NF2 loss showed 53.2% sensitivity and 100% specificity in differentiating malignant pleural mesothelioma from reactive mesothelial hyperplasia. A combination of NF2 FISH, 9p21 FISH, and BAP1 immunohistochemistry yielded greater sensitivity (100%) than that detected for either diagnostic assay alone (53.2% for NF2 FISH, 78.7% for 9p21 FISH, 70.2% for MTAP immunohistochemistry, or 57.4% for BAP1 immunohistochemistry). Thus, NF2 FISH in combination with other diagnostic assays is effective for distinguishing malignant pleural mesothelioma from reactive mesothelial hyperplasia.

Hypomorphic IL2RG mutations may lead to milder phenotypes than X-SCID, named variably as atypical X-SCID or X-CID. We report an 11-year-old boy with a novel c. 172C>T;p.(Pro58Ser) mutation in IL2RG, presenting with atypical X-SCID phenotype. We also review the growing number of hypomorphic IL2RG mutations causing atypical X-SCID. We studied the patient’s clinical phenotype, B, T, NK, and dendritic cell phenotypes, IL2RG and CD25 cell surface expression, and IL-2 target gene expression, STAT tyrosine phosphorylation, PBMC proliferation, and blast formation in response to IL-2 stimulation, as well as protein-protein interactions of the mutated IL2RG by BioID proximity labeling. The patient suffered from recurrent upper and lower respiratory tract infections, bronchiectasis, and reactive arthritis. His total lymphocyte counts have remained normal despite skewed T and B cells subpopulations, with very low numbers of plasmacytoid dendritic cells. Surface expression of IL2RG was reduced on his lymphocytes. This led to impaired STAT tyrosine phosphorylation in response to IL-2 and IL-21, reduced expression of IL-2 target genes in patient CD4+ T cells, and reduced cell proliferation in response to IL-2 stimulation. BioID proximity labeling showed aberrant interactions between mutated IL2RG and ER/Golgi proteins causing mislocalization of the mutated IL2RG to the ER/Golgi interface. In conclusion, IL2RG p.(Pro58Ser) causes X-CID. Failure of IL2RG plasma membrane targeting may lead to atypical X-SCID. We further identified another carrier of this mutation from newborn SCID screening, lost to closer scrutiny. Electronic supplementary material The online version of this article (10.1007/s10875-020-00745-2) contains supplementary material, which is available to authorized users.

Intellectual disability (ID) is one of most frequent reasons for genetic consultation. The complex molecular anatomy of ID ranges from complete chromosomal imbalances to single nucleotide variant changes occurring de novo, with thousands of genes identified. This extreme genetic heterogeneity challenges the molecular diagnosis, which mostly requires a genomic approach. CXorf56 is largely uncharacterized and was recently proposed as a candidate ID gene based on findings in a single Dutch family. Here, we describe nine cases (six males and three females) from three unrelated families. Exome sequencing and combined database analyses, identified family-specific CXorf56 variants (NM_022101.3:c.498_503del, p.(Glu167_Glu168del) and c.303_304delCTinsACCC, p.(Phe101Leufs*20)) that segregated with the ID phenotype. These variants are presumably leading to loss-of-function, which is the proposed disease mechanism. Clinically, CXorf56-related disease is a slowly progressive neurological disorder. The phenotype is more severe in hemizygote males, but might also manifests in heterozygote females, which showed skewed X-inactivation patterns in blood. Male patients might present previously unreported neurological features such as epilepsy, abnormal gait, tremor, and clonus, which extends the clinical spectrum of the disorder. In conclusion, we confirm the causative role of variants in CXorf56 for an X-linked form of intellectual disability with additional neurological features. The gene should be considered for molecular diagnostics of patients with ID, specifically when family history is suggestive of X-linked inheritance. Further work is needed to understand the role of this gene in neurodevelopment and intellectual disability.

Dent disease (DD) is a rare X-linked proximal tubulopathy associated with low molecular weight proteinuria (LMWP), hypercalciuria, nephrolithiasis and phosphoruria, which may progress to chronic kidney disease (CKD). About 60% of cases are caused by the mutation in CLCN5 gene. Recently, we identified a mutation in the sequence of homodimer of CLCN5 gene in a patient with DD. The Peripheral Blood Mononuclear Cells (PBMCs) of the patient were obtained and a line of induced pluripotent stem cells (iPSCs) was successfully generated. The iPSC line will be useful for further study of the pathogenesis and drug screening for DD.

INTRODUCTION: The acronym VATER/VACTERL refers to the rare non-random association of the following component features (CFs): vertebral defects (V), anorectal malformations (ARM) (A), cardiac anomalies (C), tracheoesophageal fistula with or without esophageal atresia (TE), renal malformations (R), and limb anomalies (L). For the clinical diagnosis the presence of at least three CFs is required, individuals presenting with only two CFs have been categorized as VATER/VACTERL-like. The majority of VATER/VACTERL individuals displays a renal phenotype. Hitherto, variants in FGF8, FOXF1, HOXD13, LPP, TRAP1, PTEN and ZIC3 have been associated with the VATER/VACTERL association; however, large-scale re-sequencing could only confirm TRAP1 and ZIC3 as VATER/VACTERL disease genes, both associated with a renal phenotype. METHODS: In this study, we performed exome sequencing in 21 individuals and their families with a renal VATER/VACTERL or VATER/VACTERL-like phenotype to identify potentially novel genetic causes. RESULTS: Exome analysis identified biallelic and X-chromosomal hemizygous potentially pathogenic variants in six individuals (29%) in B9D1, FREM1, ZNF157, SP8, ACOT9, and TTLL11, respectively. The online tool GeneMatcher revealed another individual with a variant in ZNF157. CONCLUSION: Our study suggests six biallelic and X-chromosomal hemizygous VATER/VACTERL disease gene implicating all six genes in the expression of human renal malformations.

Plasmodium vivax malaria is endemic in Mauritania. Individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency may develop acute hemolytic anemia when exposed to 8-aminoquinoline antimalarial drugs, which are indispensable for a complete cure. The prevalence of G6PD allelic variants was assessed in different ethno-linguistic groups present in Mauritania. A total of 996 blood samples (447 males and 549 females, 499 white Moors and 497 individuals of black African ancestry) were collected from febrile patients in 6 different study sites: Aleg, Atar, Kiffa, Kobeni, Nouakchott, and Rosso. The presence of the African-type G6PD A- (G202A, A376G, A542T, G680T, and T968C mutations) and the Mediterranean-type G6PD B- (C563T) variants was assessed by PCR followed by restriction fragment length polymorphism and/or DNA sequencing. The prevalence of African-type G6PD A- genotype was 3.6% (36/996), with 6.3% (28/447) of hemizygote (A-) males and 1.5% (8/549) of homozygous (A-A-) females. Forty of 549 (7.3%) women were heterozygous (AA-). The following genotypes were observed among hemizygous men and/or homozygous women: A376G/G202A (22/996, 2.2%), A376G/T968C Betica-Selma (12/996, 1.2%), and A376G/A542T Santamaria (2/996, 0.2%). The Mediterranean-type G6PD B- genotype was not observed. The prevalence rates of G6PD A- genotype in male (10/243, 4.1%) and heterozygous female (6/256, 2.3%) white Moors were lower (p &lt, 0.05) than those of males (18/204, 8.8%) and heterozygous females (34/293, 11.6%) of black African ancestry. There were only a few homozygous women among both white Moors (3/256, 1.2%) and those of black African ancestry (5/293, 1.7%). The prevalence of G6PD deficiency in Mauritania was comparable to that of neighboring countries in the Maghreb. Because of the purportedly close ethnic ties between the Mauritanian white Moors and the peoples in the Maghreb, further investigations on the possible existence of the Mediterranean-type allele are required. Moreover, a surveillance system of G6PD phenotype and/or genotype screening is warranted to establish and monitor a population-based prevalence of G6PD deficiency.

Charcot-Marie-Tooth (CMT) disease is the most common hereditary peripheral neuropathy. X-linked Charcot-Marie-Tooth disease in the GJB1 gene is known as CMTX1. We report a 14 years-old young man with walked unstably, bilateral strephenopodia, severe alopecia and paroxysmal bilateral upper limbs tremor without obvious muscle atrophy. Diagnostic whole-exome sequencing revealed a hemizygote missense mutation c.278 T > A in exon 2 of the GJB1 gene, with lysine at position 93 of the mature protein (p.M93K). This is the first CMT case with alopecia areata reported in the world.

Reducing body myopathy is a rare X-linked myopathy characterized by the presence of reducing bodies. The causative gene has been identified as FHL1. We presented with the clinical, muscle magnetic resonance imaging and genetic features of 6 unrelated Chinese patients with reducing body myopathy. We divided the patients into 2 groups according to their age at onset. In addition to limb muscle weakness, pronounced axial muscle involvement was a striking feature common to both groups. Muscle magnetic resonance imaging revealed fatty infiltration predominantly in the postero-medial muscles of the thigh and the soleus muscle of the calf, sparing the gluteus and sartorius muscles. Muscle pathology demonstrated the muscle fibres with reducing bodies distributed in small groups. Genetic analysis revealed FHL1 hemizygote variants in the 6 patients, including 4 novel and 2 reported variants. These variants were located in the LIM2 domain of FHL1 in 4 patients, but 2 located in the LIM4 domain. To the best of our knowledge, this is the first report of reducing body myopathy in the Chinese population. Our findings expand the genetic spectrum of reducing body myopathy.

In this report, we described a large Han-Chinese family which presents with various phenotypes from unaffected to manifested nystagmus in females. Infantile nystagmus (IN) is characterized by bilateral, involuntary, and periodic eyeball oscillation, occurring at birth or within the first 6 months. The most common inheritance pattern of IN is an X-linked form with incomplete penetrance among females, and the FERM domain containing 7 gene (FRMD7) is a main disease-causing gene. A combination of exome sequencing and Sanger sequencing, as well as detailed clinical examinations were performed on the Chinese IN family. An FRMD7 c.47T>C (p.Phe16Ser) variant was proposed as the disease-causing variant. Incomplete penetrance was found in females with the FRMD7 c.47T>C variant, and hemizygous male affected subjects presented more severe manifestations compared to heterozygous female affected subjects. These findings could enhance genetic counseling and antenatal diagnosis of IN.

The c-Myb gene encodes a transcription factor that regulates cell proliferation, differentiation, and apoptosis through protein-protein interaction and transcriptional regulation of signaling pathways. The protein is frequently overexpressed in human leukemias, breast cancers, and other solid tumors suggesting that it is a bona fide oncogene. c-MYB is often overexpressed by translocation in human tumors with t(6;7)(q23;q34) resulting in c-MYB-TCRβ in T cell ALL, t(X;6)(p11;q23) with c-MYB-GATA1 in acute basophilic leukemia, and t(6;9)(q22–23;p23–24) with c-MYB-NF1B in adenoid cystic carcinoma. Antisense oligonucleotides to c-MYB were developed to purge bone marrow cells to eliminate tumor cells in leukemias. Recently small molecules that inhibit c-MYB activity have been developed to disrupt its interaction with p300. The Dmp1 (cyclin D binding myb-like protein 1; Dmtf1) gene was isolated through its virtue for binding to cyclin D2. It is a transcription factor that has a Myb-like repeat for DNA binding. The Dmtf1 protein directly binds to the Arf promoter for transactivation and physically interacts with p53 to activate the p53 pathway. The gene is hemizygously deleted in 35–42% of human cancers, and is associated with longer survival. The significances of aberrant expression of c-MYB and DMTF1 proteins in human cancers and their clinical significances are discussed.

The 6-month Tg.rasH2 mouse carcinogenicity model provides an acceptable alternative to the 2-year carcinogenicity study in CD-1 mice. However, key questions related to the use of this model for testing antisense oligonucleotides (ASOs) include the similarity in the biologic response between mouse strains and the feasibility of using data from the CD-1 mouse to set doses and dose schedules for a Tg.rasH2 carcinogenicity study. To evaluate the potential strain differences, four distinct 2′- O-(2-methoxyethyl) ASOs were administered to CByB6F1 (wild type), Tg.rasH2 (hemizygous), and CD-1 mice. There were no meaningful differences in clinical signs, body weight, food consumption, or serum chemistry and hematology parameters. Histopathology evaluation indicated little to no difference in the spectrum or magnitude of changes present. The cytokine/chemokine response was also not appreciably different between the strains. This was consistent with the similarity in ASO concentration in the liver between the mouse strains tested. As the class effects of the ASOs were not meaningfully different between CD-1, CByB6F1, or Tg.rasH2 mice, data from nonclinical studies in CD-1 mice can be used for dose selection and expectation of effect in the Tg.rasH2 mouse.

Hearing impairment (HI) is a common disorder of sensorineural function with a highly heterogeneous genetic background. Although substantial progress has been made in the understanding of the genetic etiology of hereditary HI, many genes implicated in HI remain undiscovered. Via exome and Sanger sequencing of DNA samples obtained from consanguineous Pakistani families that segregate profound prelingual sensorineural HI, we identified rare homozygous missense variants in four genes (ADAMTS1, MPDZ, MVD, and SEZ6) that are likely the underlying cause of HI. Linkage analysis provided statistical evidence that these variants are associated with autosomal recessive nonsyndromic HI. In silico analysis of the mutant proteins encoded by these genes predicted structural, conformational or interaction changes. RNAseq data analysis revealed expression of these genes in the sensory epithelium of the mouse inner ear during embryonic, postnatal, and adult stages. Immunohistochemistry of the mouse cochlear tissue, further confirmed the expression of ADAMTS1, SEZ6, and MPDZ in the neurosensory hair cells of the organ of Corti, while MVD expression was more prominent in the spiral ganglion cells. Overall, supported by in silico mutant protein analysis, animal models, linkage analysis, and spatiotemporal expression profiling in the mouse inner ear, we propose four new candidate genes for HI and expand our understanding of the etiology of HI.

Sub‐heading Compound hemizygous variants in SERPINA7 gene. Background Thyroxine‐binding globulin (TBG) is encoded by SERPINA7 (OMIM. 314200) which is located on Xq22.3. SERPINA7 variants caused TBG deficiency which does not require treatment, but the decreased thyroxine may be misdiagnosed as hypothyroidism. We discovered some variants of TBG caused by alterations that differ from previously reported. Materials and Methods In this study, we enrolled 32 subjects from 10 families and sequenced the SERPINA7 genes of TBG‐deficient subjects. Then, variants were analyzed to assess their effect on TBG expression and secretion. Bioinformatics database, protein structure, and dynamics simulation were used to evaluate the deleterious effects. Finally, we identified 2 novel and 4 known variants, and found 26 of 30 subjects carried the p.L303F. The DynaMut predictions indicated the variants (p.E91K, p.I92T, p.R294C, and p.L303F) exhibited decreased stability. Conclusion Analyses revealed the p.L303F change the protein stability and flexibility, and it had an impact on the function of TBG, but when coexisted with other variants it might change the conformational structure of the protein and aggravate the damage to the protein. We speculated that the existence of a higher number of variants resulted in lower TBG secretion., The p.L303F that previouly identified as a variant had an impact on the function and stability of TBG, and when coexisted with other variants it might change the conformational structure of the protein and aggravate the damage to the protein. The existence of a higher number of variants resulted in lower TBG secretion.

Background and Aims Mutations in XIAP can lead to the development of treatment-refractory severe paediatric Crohn’s disease [CD], for which haematopoietic stem cell transplantation is the primary therapeutic option. The interpretation of variants of uncertain significance [VUSs] in XIAP needs to be scrutinized. Methods Targeted next-generation sequencing was performed for 33 male paediatric patients with refractory CD admitted at a tertiary referral hospital. To obtain functional data, biomolecular cell assays and supercomputing molecular dynamics simulations were performed. Results Nine unrelated male patients harboured hemizygous XIAP variants. Four known pathogenic variants and one novel pathogenic variant [p.Lys168Serfs*12] were identified in five patients, and two novel VUSs [p.Gly205del and p.Pro260Ser] and one known VUS [p.Glu350del] were identified in the remaining four. Among children with VUSs, only the subject with p.Gly205del exhibited defective NOD2 signalling. Using molecular dynamics simulation, we determined that the altered backbone torsional energy of C203 in XIAP of p.G205del was ~2 kcal/mol, suggesting loss of zinc binding in the mutant XIAP protein and poor coordination between the mutant XIAP and RIP2 proteins. Elevated auto-ubiquitination of zinc-depleted p.G205del XIAP protein resulted in XIAP protein deficiency. Conclusion A high prevalence of XIAP deficiency was noted among children with refractory CD. Advanced functional studies decreased the subjectivity in the case-level interpretation of XIAP VUSs and directed consideration of haematopoietic stem cell transplantation.

Background Ambiguous or atypical phenotypes can make a definite diagnosis of primary immunodeficiency diseases based on biochemical indices alone challenging. Further, mortality in early life because of infections in patients with these conditions supports the use of genetic tests to facilitate rapid and accurate diagnoses. Methods Genetic and clinical analyses of three unrelated Chinese children with clinical manifestations of recurrent infections, who were considered to have primary immunodeficiency diseases, were conducted. Patient clinical features and serum immunological indices were recorded. Next‐generation sequencing was used to screen for suspected pathogenic variants. Family co‐segregation and in silico analysis were conducted to evaluate the pathogenicity of identified variants, following the American College of Medical Genetics and Genomics guidance. Results All three patients were found to have predominant antibody defects. Sequencing analysis revealed that one had two compound heterozygous variants, c.255C>A and c.295C>T, in the autosomal gene, activation‐induced cytidine deaminase (AICDA). The other two patients were each hemizygous for the variants c.1185G>A and c.82C>T in the Bruton's tyrosine kinase (BTK) gene on the X chromosome. In silico analysis revealed that identified substituted amino acids were highly conserved and predicted to cause structural and functional damage to the proteins. Conclusion Four pathogenic variants in AICDA and BTK were confirmed to cause different forms of hyper‐IgM syndrome type 2 (HIGM2) and X‐linked agammaglobulinemia (XLA); two were novel mutations that have never been reported previously. This is the first report of HIGM2 caused by AICDA deficiency in a patient from the Chinese mainland., In this paper, comprehensive clinical characteristics of the PIDDs patients are recorded, which will give more evidence for the diagnosis of the two diseases. Two novel mutations have never been reported before were identified, which will enrich the worldwide spectrum of HIGM2 and XLA. Moreover, this is the first report of HIGM2 caused by AICDA deficiency in the Chinese mainland cohorts, which will set a good example for similar cases in the future.

Xq25 microduplication syndrome is a recognized syndrome presenting intellectual disability and distinctive facial appearance. We generated an induced pluripotent stem cell (iPSC) line from peripheral blood mononuclear cells (PBMCs) of an 8-year-old boy with Xq25 Microduplication Syndrome carrying a 1.3 Mb hemizygote duplication at chrXq25. The iPSCs expressed pluripotency markers, free of genomically integrated episomal plasmids, with normal karyotype and three layers’ differentiation potential in vitro.