Heterozygous Nonsense Mutation Research Articles

Plasma cell deficiency in human subjects with heterozygous mutations in Sec61 translocon alpha 1 subunit (SEC61A1)

Primary antibody deficiencies (PADs) are the most frequent primary immunodeficiencies in human subjects. The genetic causes of PADs are largely unknown. Sec61 translocon alpha 1 subunit (SEC61A1) is the major subunit of the Sec61 complex, which is the main polypeptide-conducting channel in the endoplasmic reticulum membrane. SEC61A1 is a target gene of spliced X-box binding protein 1 and strongly induced during plasma cell (PC) differentiation. We identified a novel genetic defect and studied its pathologic mechanism in 11 patients from 2 unrelated families with PADs. Whole-exome and targeted sequencing were conducted to identify novel genetic mutations. Functional studies were carried out exvivo in primary cells of patients and invitro in different cell lines to assess the effect of SEC61A1 mutations on B-cell differentiation and survival. We investigated 2 families with patients with hypogammaglobulinemia, severe recurrent respiratory tract infections, and normal peripheral B- and T-cell subpopulations. On invitro stimulation, B cells showed an intrinsic deficiency to develop into PCs. Genetic analysis and targeted sequencing identified novel heterozygous missense (c.254T>A, p.V85D) and nonsense (c.1325G>T, p.E381*) mutations in SEC61A1, segregating with the disease phenotype. SEC61A1-V85D was deficient in cotranslational protein translocation, and it disturbed the cellular calcium homeostasis in HeLa cells. Moreover, SEC61A1-V85D triggered the terminal unfolded protein response in multiple myeloma cell lines. We describe a monogenic defect leading to a specific PC deficiency in human subjects, expanding ourknowledge about the pathogenesis of antibody deficiencies.

Identification of a novel de novo ANK1 R1426* nonsense mutation in a Chinese family with hereditary spherocytosis by NGS.

Hereditary spherocytosis (HS) is an inherited heterogeneous hemolytic anemia, characterized by the presence of spherical-shaped erythrocytes on the peripheral blood smear, and the clinical manifestation ranges from asymptomatic to severely anemic, and transfusion-dependent patients. Mutations in at least five genes (ANK1, EPB42, SLC4A1, SPTA1, and SPTB) have been identified so far, and mutations of ANK1 gene are responsible for the majority of all HS cases. In this study, targeted next generation sequencing (NGS) was applied to identify a novel de novo ANK1 c.4276C>T (p.R1426*) nonsense mutation in a Chinese family with a patient of HS who was diagnosed clinically with only 10% spherical-shaped erythrocytes in the peripheral blood and received splenectomy. Sanger sequencing further confirmed that only the patient carried heterozygous ANK1 c.4276C>T nonsense mutation, while none of his parents or his young brother carried this mutation. Moreover, consistent with the genetic findings, the anemia was ameliorated after splenectomy. RBCs increased from 2.74 × 1012/L pre-surgery to 4.76 × 1012/L one month post-surgery, and hemoglobin increased from 66g/L to 126g/L respectively. This is the first report of ANK1 c.4276C>T (p.R1426*) heterozygous nonsense mutation responsible for HS. Our results also demonstrate that targeted NGS may provide a powerful approach for rapid genetic test of HS.

HSP and deafness: Neurocristopathy caused by a novel mosaic SOX10 mutation.

Objective:To identify the underlying genetic cause in 2 sisters affected with progressive lower extremity spasticity, neuropathy, and early-onset deafness.Methods:Whole-exome sequencing was performed, and segregation testing of variants was investigated using targeted Sanger sequencing. An inherited paternal mosaic mutation was further evaluated through quantitative analysis of the ratio of mutant vs wild-type allele in genomic DNA from various tissues, including blood, dermal fibroblasts, and saliva.Results:A novel heterozygous nonsense mutation (c.1140C>A; p.Y380X) in SOX10 was identified in the affected sisters. Paternal mosaicism was suspected based on a small chromatogram peak, which was less than the heterozygous peak of the mutated allele. Consistent with mosaicism, the mosaic paternal samples had notable variability in the ratio of mutant vs wild-type allele in various tissues (compared with the fully heterozygous daughter), with the highest paternal mutant levels in saliva (32.7%) and lowest in dermal fibroblasts (13.9%). Targeted clinical re-examination of the father revealed a sensorimotor neuropathy that was previously clinically unrecognized.Conclusions:These findings expand the phenotypic spectrum of SOX10-related neurocristopathy. Mutations in SOX10 should be considered in patients presenting with a complicated form of hereditary spastic paraplegia that includes neuropathy and deafness. Diagnostic workup may be complicated, as SOX10 mutations can present in a mosaic state, with a mild clinical manifestation.

Propionic Academia: Exome Sequencing Identified Novel Mutation in Exon 12 of PCCA Gene

Propionic acidemia (PA) is a rare autosomal recessive genetic disorder resulting from propionyl-coenzyme A carboxylase deficiency. This mitochondrial enzyme is a heterocopolymer of two subunits a and b that are encoded by PCCA and PCCB genes, respectively. The phenotypes of PCCA mutations are most varied ranging from mild to lethal within a few days after birth because there is great heterogeneity in the mutations of PCCA, while PCCB bears only a limited number of mutations. Deep resequencing of all human genes could potentially identify allelic variants involved in a given rare monogenic disease. Whole exome sequencing (WES) is well justified as an efficient strategy to search for mutations in rare Mendelian disorders. Here, we analyzed an Iranian couple with the history of having a child who died probably due to PA by WES. We could report a novel heterozygous non-sense mutation (p.Y380X) in exon 12 of the PCCA gene, resulting in the protein truncation. We could find that both of parents are carrier for a heterozygous novel non-sense mutation in PCCA gene. We showed that WES in parents, along with appropriate filtering against public SNP databases, is sufficient to identify carriers for a known monogenic disorder, PA, in a consanguineous family. J Med Cases. 2017;8(4):111-113 doi: https://doi.org/10.14740/jmc2743e

Clinical Auditory Phenotypes Associated with GATA3 Gene Mutations in Familial Hypoparathyroidism-deafness-renal Dysplasia Syndrome.

Background:Hypoparathyroidism-deafness-renal dysplasia (HDR) syndrome is an autosomal dominant disorder primarily caused by haploinsufficiency of GATA binding protein 3 (GATA3) gene mutations, and hearing loss is the most frequent phenotypic feature. This study aimed at identifying the causative gene mutation for a three-generation Chinese family with HDR syndrome and analyzing auditory phenotypes in all familial HDR syndrome cases.Methods:Three affected family members underwent otologic examinations, biochemistry tests, and other clinical evaluations. Targeted genes capture combining next-generation sequencing was performed within the family. Sanger sequencing was used to confirm the causative mutation. The auditory phenotypes of all reported familial HDR syndrome cases analyzed were provided.Results:In Chinese family 7121, a heterozygous nonsense mutation c.826C>T (p.R276*) was identified in GATA3. All the three affected members suffered from sensorineural deafness and hypocalcemia; however, renal dysplasia only appeared in the youngest patient. Furthermore, an overview of thirty HDR syndrome families with corresponding GATA3 mutations revealed that hearing impairment occurred earlier in the younger generation in at least nine familial cases (30%) and two thirds of them were found to carry premature stop mutations.Conclusions:This study highlights the phenotypic heterogeneity of HDR and points to a possible genetic anticipation in patients with HDR, which needs to be further investigated.

A Novel CCM1/KRIT1 Heterozygous Nonsense Mutation (c.1864C>T) Associated with Familial Cerebral Cavernous Malformation: a Genetic Insight from an 8-Year Continuous Observational Study.

Cerebral cavernous malformation (CCM) is a congenital vascular abnormality that predominantly affects the central nervous system, but that sometimes encroaches other vital tissues, including the retina, skin, and even liver. The familial form of CCM (FCCM) is considered to be an autosomal dominant disease with incomplete penetrance and variable expression, which is often attributed to mutations in three genes: CCM1, CCM2, and CCM3. We screened a Chinese family diagnosed with FCCM by using Sanger sequencing. A 29-year-old male proband with cutaneous angiomas was pathologically diagnosed but presented with an atypical form of CCM as revealed by magnetic resonance imaging (MRI) findings, prompting further clinical evaluation and genetic analyses of him and his immediate family. We performed continuous observation over an 8-year period using MRI gradient echo imaging and susceptibility-weighted imaging of these individuals. Sanger sequencing of the CCM1, CCM2, and CCM3 genes identified a novel heterozygous nonsense nucleotide transition (c.1864C>T; p.Gln622X) in exon 17 of the CCM1/KRIT1 gene; this mutation was predicted to cause a premature stop codon (TAG) at nucleotides 1864 to 1866 to generate a truncated Krev interaction trapped 1 (Krit1) protein of 621 amino acids. During this long-term observational study, one of the enrolled family members with neurological deficits progressed to a stage indicative of brain surgery. This study provides a new CCM gene mutation profile, which highlights the significance of genetic counseling for individuals suspected of having this condition.

POLR2C Mutations Are Associated With Primary Ovarian Insufficiency in Women.

Context:Primary ovarian insufficiency (POI) results from a premature loss of oocytes, causing infertility and early menopause. The etiology of POI remains unknown in a majority of cases.Objective:To identify candidate genes in families affected by POI.Design:This was a family-based genetic study.Setting:The study was performed at two academic institutions.Patients and Other Participants:A family with four generations of women affected by POI (n = 5). Four of these women, three with an associated autoimmune diagnosis, were studied. The controls (n = 387) were recruited for health in old age.Intervention:Whole-genome sequencing was performed.Main Outcome Measure:Candidate genes were identified by comparing gene mutations in three family members and 387 control subjects analyzed simultaneously using the pedigree Variant Annotation, Analysis and Search Tool. Data were also compared with that in publicly available databases.Results:We identified a heterozygous nonsense mutation in a subunit of RNA polymerase II (POLR2C) that synthesizes messenger RNA. A rare sequence variant in POLR2C was also identified in one of 96 women with sporadic POI. POLR2C expression was decreased in the proband compared with women with POI from another cause. Knockdown in an embryonic carcinoma cell line resulted in decreased protein production and impaired cell proliferation.Conclusions:These data support a role for RNA polymerase II mutations as candidates in the etiology of POI. The current data also support results from genome-wide association studies that hypothesize a role for RNA polymerase II subunits in age at menopause in the population.

Three-Year Follow-Up of High-Dose Ubiquinol Supplementation in a Case of Familial Multiple System Atrophy with Compound Heterozygous COQ2 Mutations

We report a 3-year follow-up of high-dose ubiquinol supplementation in a case of familial multiple system atrophy (MSA) with compound heterozygous nonsense (R387X) and missense (V393A) mutations in COQ2. A high-dose ubiquinol supplementation substantially increased total coenzyme Q10 levels in cerebrospinal fluid as well as in plasma. The patient was at the advanced stage of MSA, and the various scores of clinical rating scales remained stable without changes during the 3 years. The cerebral metabolic ratio of oxygen measured by 15O2 PET, however, increased by approximately 30% after administration of ubiquinol, suggesting that ubiquinol can improve mitochondrial oxidative metabolism in the brain. It also suggests the therapeutic potential of ubiquinol for patients with MSA with COQ2 mutations. Further clinical trials of administration of high-dose ubiquinol to MSA patients are warranted.

Diagnosis for choroideremia in a large Chinese pedigree by next‑generation sequencing (NGS) and non‑invasive prenatal testing (NIPT).

To develop an effective strategy to isolate and use cell-free fetal DNA (cffDNA) for the combined use of next-generation sequencing (NGS) for diagnosing choroideremia and non-invasive prenatal testing (NIPT) for Y chromosome determination, a large Chinese family with an X-linked recessive disease, choroideremia, was recruited. Cell-free DNA was extracted from maternal plasma, and SRY polymerase chain reaction amplification was performed using NIPT. Sanger sequencing was subsequently used for fetal amniotic fluid DNA verification. A nonsense mutation (c.C799T:p.R267X) of the CHM gene on the X chromosome of the proband (IV:7) and another 5 males with choroideremia were detected, while 3 female carriers with no symptoms were also identified. The fetus (VI:7) was identified as female from the cffDNA, and the same heterozygous nonsense mutation present in her mother was also confirmed. At one and a half years of age, the female baby did not present with any associated symptoms of choroideremia. Therefore, cffDNA was successfully used for the combined use of NGS for diagnosing choroideremia in a large Chinese pedigree, and NIPT for Y chromosome determination. This approach should result in a markedly increased use of prenatal diagnosis and improvement, and more sophisticated clinical management of diseases in China and other developing countries. The establishment of a highly accurate method for prenatal gene diagnosis will allow for more reliable gene diagnosis, improved genetic counseling, and personalized clinical management of our patients.

Liver Failure as the Presentation of Ornithine Transcarbamylase Deficiency in a 13-Month-Old Female.

Ornithine transcarbamylase deficiency (OTCD) is an X-linked urea cycle disorder with variable expressivity in heterozygous females. While liver function testing is often abnormal in patients with OTCD, liver failure is uncommon on presentation. A 13-month-old female with no significant past medical history presented with irritability, right arm weakness, and decreased appetite. Initial workup revealed hepatic dysfunction with an INR of 3.4, ammonia level of 75μmol/L, and abnormal brain MRI with gyral edema with restricted diffusion, and patchy signal abnormality in basal ganglia. The MRI findings led to a putative diagnosis of acute disseminated encephalomyelitis prompting corticosteroid treatment. As steroid treatment was begun, she developed significant hepatocellular dysfunction with ALT 2,222U/L, AST 630U/L, prolonged INR, and elevated ammonia (213μmol/L). Neurologic signs resolved and her ammonia level decreased (43μmol/L) without further intervention; however, she had ongoing acute liver failure with coagulopathy and episodic irritability, managed as seronegative autoimmune hepatitis with partial response to corticosteroid therapy. At 18months of age she presented with severe irritability with markedly increased ammonia (417μmol/L). Plasma amino acids obtained several days prior to this acute episode demonstrated elevation in glutamine (2,725μmol/L) and alanine (1,459μmol/L). Biochemical testing demonstrated elevation of urine orotic acid (>240.6mmol/mol creatinine). Genetic testing confirmed a heterozygous nonsense mutation in the OTC gene (c.958C>T, R320X). After treatment with ammonia scavengers and a protein-restricted diet, hepatic function normalized and irritability resolved. The diagnosis of a urea cycle disorder should be considered in patients with unexplained hepatic dysfunction.

PCTP (Phosphatidylcholine Transfer Protein) is Regulated By RUNX1 in Platelets/Megakaryocytes and is Associated with Adverse Cardiovascular Events

Transcription factor (TF) mutations are increasingly recognized to play a major role in inherited platelet abnormalities. RUNX1, a major hematopoietic TF, acts in a combinatorial manner with other TFs to regulate numerous megakaryocyte (MK)/platelet genes. Human RUNX1 haplodeficiency is associated with thrombocytopenia, platelet function defects, and increased leukemia risk. We have described a patient with multiple abnormalities in platelet aggregation and secretion responses with a heterozygous RUNX1 nonsense mutation (Sun et al Blood 2004; 103; 948-54). Transcript expression profiling of patient platelets (Sun et al J Thromb Haemost 2007; 5:146-54)showed several genes were significantly downregulated, including myosin light chain (MYL9), platelet factor 4 (PF4), protein kinase C-θ (PRCKQ), and 12-lipoxygenase (ALOX12); these have been shown by us to be regulated by RUNX1. The profiling data also showed 10-fold downregulation of phosphatidylcholine transfer protein (PCTP) gene (fold change ratio 0.09, p=0.02) in the patient compared with normal controls. PCTP regulates the intermembrane transfer of phosphatidylcholine (PC), a major plasma membrane phospholipid. Platelet PCTP expression is associated with increased platelet aggregation and calcium mobilization upon activation of protease-activated receptor 4 (PAR4) thrombin receptors in black subjects as compared to white subjects (Edelstein et al Nat Med 2013; 19:1609-16). Pharmacologic inhibition of PCTP decreased platelet aggregation in response to PAR4 agonist and siRNA knockdown of PCTP in megakaryocytic cells blunted calcium mobilization induced by PAR4 (Edelstein et al Nat Med 2013; 19:1609-16). Little is known regarding the regulation of PCTP in MKs/platelets and its role in cardiovascular events.Based on the decreased platelet PCTP expression in our patient, we pursued the hypothesis that PCTP is regulated by RUNX1 and contributes to cardiovascular events. Corrected total cellular immunofluorescence with anti-PCTP antibody showed significantly reduced platelet PCTP expression by 58% in our patient compared to a normal control. In silico analysis revealed 5 RUNX1 consensus binding sites up to ~ 1 kb of the PCTP 5’ upstream region from ATG. To assess for interaction of RUNX1 with the PCTP promoter, chromatin immunoprecipitation (ChIP) assay with anti-RUNX1 antibody was performed using human erythroid leukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 48 hours to induce megakaryocytic transformation. The ChIP studies showed RUNX1 binding to PCTP chromatin in the regions encompassing RUNX1 binding site 1 (-345/-340), site 3 (-632/-627), and encompassing sites 4 and 5 (-974/-969, -997/-992). Electrophoretic mobility shift assay (EMSA) using PMA-treated HEL cell nuclear extracts showed RUNX1 binding to DNA probes (28-37 bp) containing site 1 (-345/-340) and both sites 4 and 5 (-974/-969, -997/-992). PCTP mRNA and protein expression were increased with RUNX1 overexpression and reduced with RUNX1 knockdown in HEL cells, indicating that PCTP is regulated by RUNX1. To assess the clinical relevance of the findings, the relationship between RUNX1 and PCTP in peripheral blood RNA, and PCTP and death or myocardial infarction (MI) events were assessed in two separate patient cohorts (n = 587 total patients) with cardiovascular disease. RUNX1 is transcribed from two alternate promoters (P1 and P2) resulting in different isoforms. In both patient cohorts, there was strong correlation between RUNX1 and PCTP expression in a promoter specific manner. RUNX1 P1 probe sets were strongly and inversely correlated with PCTP expression (p < 0.0001), while the P2 probe sets were not. PCTP expression was associated with death or MI in both patient cohorts (odds ratio 2.1, 95% CI [1.61-2.95], P-value < 0.0001) independent of age, sex, race, platelet count, and cardiovascular risk factors.Conclusions: Our results provide evidence that PCTP is regulated by RUNX1 (potentially in a promoter specific manner), and that PCTP expression is associated with death or myocardial infarction in patients with cardiovascular disease. RUNX1 regulation of PCTP may play a role in the pathogenesis of platelet-mediated cardiovascular events. DisclosuresNo relevant conflicts of interest to declare.

Congenital Mirror Movements Due to RAD51: Cosegregation with a Nonsense Mutation in a Norwegian Pedigree and Review of the Literature.

BackgroundAutosomal dominant congenital mirror movements (CMM) is a neurodevelopmental disorder characterized by early onset involuntary movements of one side of the body that mirror intentional movements on the contralateral side; these persist throughout life in the absence of other neurological symptoms. The main culprit genes responsible for this condition are RAD51 and DCC. This condition has only been reported in a few families, and the molecular mechanisms linking RAD51 mutations and mirror movements (MM) are poorly understood.MethodsWe collected demographic, clinical, and genetic data of a new family with CMM due to a truncating mutation of RAD51. We reviewed the literature to identify all reported patients with CMM due to RAD51 mutations.ResultsWe identified a heterozygous nonsense mutation c.760C>T (p.Arg254*) in eight subjects: four with obvious and disabling MM, and four with a mild phenotype. Including our new family, we identified 32 patients from 6 families with CMM linked to RAD51 variants.DiscussionOur findings further support the involvement of RAD51 in CMM pathogenesis. Possible molecular mechanisms involved in CMM pathogenesis are discussed.

Identification of Heterozygous Single- and Multi-exon Deletions in IL7R by Whole Exome Sequencing

PurposeWe aimed to achieve a retrospective molecular diagnosis by applying state-of-the-art genomic sequencing methods to past patients with T-B+NK+ severe combined immunodeficiency (SCID). We included identification of copy number variations (CNVs) by whole exome sequencing (WES) using the CNV calling method ExomeDepth to detect gene alterations for which routine Sanger sequencing analysis is not suitable, such as large heterozygous deletions.MethodsOf a total of 12 undiagnosed patients with T-B+NK+ SCID, we analyzed eight probands by WES, using GATK to detect single nucleotide variants (SNVs) and small insertions and deletions (INDELs) and ExomeDepth to detect CNVs.ResultsWe found heterozygous single- or multi-exon deletions in IL7R, a known disease gene for autosomal recessive T-B+NK+ SCID, in four families (seven patients). In three families (five patients), these deletions coexisted with a heterozygous splice site or nonsense mutation elsewhere in the same gene, consistent with compound heterozygosity. In our cohort, about a quarter of T-B+NK+ SCID patients (26%) had such compound heterozygous IL7R deletions.ConclusionsWe show that heterozygous IL7R exon deletions are common in T-B+NK+ SCID and are detectable by WES. They should be considered if Sanger sequencing fails to detect homozygous or compound heterozygous IL7R SNVs or INDELs.

A SDHC Founder Mutation Causes Paragangliomas (PGLs) in the French Canadians: New Insights on the SDHC-Related PGL.

Background:More than 40% of patients with paragangliomas (PGLs) harbor a germline mutation of the known PGL susceptibility genes, mainly in the SDHB or SDHD genes.Objective:The objective of the study was to characterize the genetic background of the French Canadian (FC) patients with PGLs and provide new clinical and paraclinical insights on SDHC-related PGLs.Methods:Genetic testing has been offered to FC patients affected with PGLs followed up at the adrenal genetics clinic at Centre hospitalier de l'Université de Montréal. After genetic counseling, 29 FC patients consented for PGL genetic testing.Results:Thirteen of 29 patients (44.8%) carried a germline mutation. The same heterozygous nonsense mutation at codon 133 of exon 5 of the SDHC gene (c.397C>T, p.[Arg133Ter]) was found in nine patients, representing 69.2% of the patients having a germline mutation. Seventy percent of these patients had head and neck PGLs. Twenty percent had multiple and 30% had malignant PGLs. We traced back the ascending genealogy of 10 index cases (nine patients from our cohort and one patient referred to us) and found that this mutation was most probably introduced in Nouvelle France by a couple of French settlers who established themselves in the 17th century.Conclusions:We found that 31% of the PGLs in the French Canadian can be explained by the SDHC mutation (c.397C>T, p.[Arg133Ter]). The dominance of the SDHC mutation is unique to the FCs and is most likely due to a French founder effect. SDHC gene analysis should be prioritized in FC patients with PGL.

Amelogenesis Imperfecta

Amelogenesis imperfecta (AI) is a clinically and genetically heterogeneous group of diseases characterized by enamel defects. The authors have identified a large consanguineous Moroccan family segregating different clinical subtypes of hypoplastic and hypomineralized AI in different individuals within the family. Using targeted next-generation sequencing, the authors identified a novel heterozygous nonsense mutation in COL17A1 (c.1873C>T, p.R625*) segregating with hypoplastic AI and a novel homozygous 8-bp deletion in C4orf26 (c.39_46del, p.Cys14Glyfs*18) segregating with hypomineralized-hypoplastic AI in this family. This study highlights the phenotypic and genotypic heterogeneity of AI that can exist even within a single consanguineous family. Furthermore, the identification of novel mutations in COL17A1 and C4orf26 and their correlation with distinct AI phenotypes can contribute to a better understanding of the pathophysiology of AI and the contribution of these genes to amelogenesis.

A novel nonsense mutation in the NOG gene causes familial NOG-related symphalangism spectrum disorder.

The human noggin (NOG) gene is responsible for a broad spectrum of clinical manifestations of NOG-related symphalangism spectrum disorder (NOG-SSD), which include proximal symphalangism, multiple synostoses, stapes ankylosis with broad thumbs (SABTT), tarsal–carpal coalition syndrome, and brachydactyly type B2. Some of these disorders exhibit phenotypes associated with congenital stapes ankylosis. In the present study, we describe a Japanese pedigree with dactylosymphysis and conductive hearing loss due to congenital stapes ankylosis. The range of motion in her elbow joint was also restricted. The family showed multiple clinical features and was diagnosed with SABTT. Sanger sequencing analysis of the NOG gene in the family members revealed a novel heterozygous nonsense mutation (c.397A>T; p.K133*). In the family, the prevalence of dactylosymphysis and hyperopia was 100% while that of stapes ankylosis was less than 100%. Stapes surgery using a CO2 laser led to a significant improvement of the conductive hearing loss. This novel mutation expands our understanding of NOG-SSD from clinical and genetic perspectives.

Gene mutational analyses of the cathepsin C gene in families with Papillon-Lefèvre syndrome

This study aims to investigate the gene mutational characteristics of cathepsin C (CTSC) gene in a Chinese patient with Papillon-Lefèvre syndrome (PLS), then further confirm the genetic basis for the phenotype of PLS, and obtain genetic information that can be used as guide in the diagnosis and treatment of PLS. With their consent, peripheral blood samples were obtained from the proband and his family members (his parents and older sister) for genomic DNA extraction. The coding region and exon/intron boundaries of the CTSC gene were amplified and sequenced using poly-merase chain reaction and direct sequencing of DNA. Compound heterozygous mutations of CTSC gene were iden-tified in the patient. The proband carries one heterozygous nonsense mutation c.754C>T in exon 5 and one heterozygous missense mutation c.1040A>G in exon 7. Both parents were heterozygous carriers without the clinical symptoms of PLS. None of the mutations were detected in the proband's sister. The study proves that mutations of CTSC gene are responsible for the phenotype of Papillon-Lefèvre syndrome. .

BCL11A Haploinsufficiency Causes an Intellectual Disability Syndrome and Dysregulates Transcription

Intellectual disability (ID) is a common condition with considerable genetic heterogeneity. Next-generation sequencing of large cohorts has identified an increasing number of genes implicated in ID, but their roles in neurodevelopment remain largely unexplored. Here we report an ID syndrome caused by de novo heterozygous missense, nonsense, and frameshift mutations in BCL11A, encoding a transcription factor that is a putative member of the BAF swi/snf chromatin-remodeling complex. Using a comprehensive integrated approach to ID disease modeling, involving human cellular analyses coupled to mouse behavioral, neuroanatomical, and molecular phenotyping, we provide multiple lines of functional evidence for phenotypic effects. The etiological missense variants cluster in the amino-terminal region of human BCL11A, and we demonstrate that they all disrupt its localization, dimerization, and transcriptional regulatory activity, consistent with a loss of function. We show that Bcl11a haploinsufficiency in mice causes impaired cognition, abnormal social behavior, and microcephaly in accordance with the human phenotype. Furthermore, we identify shared aberrant transcriptional profiles in the cortex and hippocampus of these mouse models. Thus, our work implicates BCL11A haploinsufficiency in neurodevelopmental disorders and defines additional targets regulated by this gene, with broad relevance for our understanding of ID and related syndromes.

Rapid molecular diagnostics of severe primary immunodeficiency determined by using targeted next-generation sequencing

Primary immunodeficiency diseases (PIDDs) are inherited disorders of the immune system. The most severe form, severe combined immunodeficiency (SCID), presents with profound deficiencies of Tcells, Bcells, or both at birth. If not treated promptly, affected patients usually do not live beyond infancy because of infections. Genetic heterogeneity of SCID frequently delays the diagnosis; a specific diagnosis is crucial for life-saving treatment and optimal management. We developed a next-generation sequencing (NGS)-based multigene-targeted panel for SCID and other severe PIDDs requiring rapid therapeutic actions in a clinical laboratory setting. The target gene capture/NGS assay provides an average read depth of approximately 1000×. The deep coverage facilitates simultaneous detection of single nucleotide variants and exonic copy number variants in one comprehensive assessment. Exons with insufficient coverage (<20× read depth) or high sequence homology (pseudogenes) are complemented by amplicon-based sequencing with specific primers to ensure 100% coverage of all targeted regions. Analysis of 20 patient samples with low T-cell receptor excision circle numbers on newborn screening or a positive family history or clinical suspicion of SCID or other severe PIDD identified deleterious mutations in 14 of them. Identified pathogenic variants included both single nucleotide variants and exonic copy number variants, such as hemizygous nonsense, frameshift, and missense changes in IL2RG; compound heterozygous changes in ATM, RAG1, and CIITA; homozygous changes in DCLRE1C and IL7R; and a heterozygous nonsense mutation in CHD7. High-throughput deep sequencing analysis with complete clinical validation greatly increases the diagnostic yield of severe primary immunodeficiency. Establishing a molecular diagnosis enables early immune reconstitution through prompt therapeutic intervention and guides management for improved long-term quality of life.

Deletion of 14-3-3σ sensitizes mice to DMBA/TPA-induced papillomatosis.

The p53-inducible cell cycle regulator 14-3-3σ exhibits tumor suppressive functions and is highly expressed in differentiating layers of the epidermis and hair follicles. 14-3-3σ/SFN/stratifin is frequently silenced in human epithelial cancers, and experimental down-regulation of 14-3-3σ expression immortalizes primary human keratinocytes. In the repeated-epilation (ER) mouse model, a heterozygous nonsense mutation of 14-3-3σ causes repeated hair-loss, hyper-proliferative epidermis, and spontaneous development of papillomas and squamous cell carcinomas in aging mice. Therefore, loss of 14-3-3σ function might contribute to epithelial tumor development. Here, we generated mice with loxP sites surrounding the single 14-3-3σ exon which allowed Cre-mediated deletion of the gene. 14-3-3σ-deficient mice are viable, but demonstrate a permanently disheveled fur. However, histological analyses of the skin did not reveal obvious defects in the hair follicles or the epidermis. Deletion of 14-3-3σ did not enhance spontaneous epidermal tumor development, whereas it increased the frequency and size of DMBA/TPA-induced papillomas. In conclusion, 14-3-3σ is dispensable for normal epidermal homeostasis but critical for suppression of chemically-induced skin carcinogenesis. In addition, these results suggest that the ER mutation of 14-3-3σ is not equivalent to loss of 14-3-3σ, but may represent a gain-of-function variant, which does not reflect the organismal function of wild-type 14-3-3σ.