Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies.

The BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Because there is no common genetic alteration causing resistance to venetoclax in chronic lymphocytic leukemia (CLL) and B-cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole-exome sequencing, methylated DNA immunoprecipitation sequencing, and genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter that is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity toward venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher oxidative phosphorylation and adenosine triphosphate production, resembling the metabolic phenotype that is seen upon venetoclax resistance. Although PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity toward both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter's syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive diffuse large B-cell lymphoma in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance.

Genome-wide linkage and sequence analysis challenge CCDC66 as a human retinal dystrophy candidate gene and support a distinct NMNAT1-related fundus phenotype.

To uncover the genotype underlying early-onset cone-rod dystrophy and central nummular macular atrophic lesion in 2 siblings from an endogamous Arab family, we performed targeted next-generation sequencing (NGS) of 44 retinal dystrophy genes, whole-exome sequencing (WES) and genome-wide linkage analysis. Targeted NGS and WES in the index patient highlighted 2 homozygous variants, a CCDC66 frameshift deletion and a novel missense NMNAT1 variant, c.500G>A (p.Asn167Ser). Linkage and segregation analysis excluded the CCDC66 variant and confirmed the NMNAT1 mutation. Biallelic NMNAT1 mutations cause Leber congenital amaurosis with a central nummular macular atrophic lesion (LCA9). The NMNAT1 mutation reported here underlied cone-rod dystrophy rather than LCA but the fundus lesion was compatible with that of LCA9 patients, highlighting that such a fundus appearance should raise suspicion for biallelic mutations in NMNAT1 when in the context of any retinal dystrophy. Although Ccdc66 mutations have been proposed to cause retinal disease in dogs, our results and public databases challenge CCDC66 as a candidate gene for human retinal dystrophy.

Genetic heterogeneity in Pakistani microcephaly families revisited.

Autosomal recessive primary microcephaly (MCPH) is a rare and heterogeneous genetic disorder characterized by reduced head circumference, low cognitive prowess and, in general, architecturally normal brains. As many as 14 different loci have already been mapped. We recruited 35 MCPH families in Pakistan and could identify the genetic cause of the disease in 31 of them. Using homozygosity mapping complemented with whole-exome, gene panel or Sanger sequencing, we identified 12 novel mutations in 3 known MCPH-associated genes - 9 in ASPM, 2 in MCPH1 and 1 in CDK5RAP2. The 2 MCPH1 mutations were homozygous microdeletions of 164,250 and 577,594 bp, respectively, for which we were able to map the exact breakpoints. We also identified four known mutations - three in ASPM and one in WDR62. The latter was initially deemed to be a missense mutation but we demonstrate here that it affects splicing. As to ASPM, as many as 17 out of 27 MCPH5 families that we ascertained in our sample were found to carry the previously reported founder mutation p.Trp1326*. This study adds to the mutational spectra of four known MCPH-associated genes and updates our knowledge about the genetic heterogeneity of MCPH in the Pakistani population considering its ethnic diversity.

Morphological alterations in two siblings with autosomal recessive congenital ichthyosis associated with CYP4F22 mutations.

Autosomal recessive congenital ichthyosis (ARCI) caused by mutations in CYP4F22 is very rare. CyP4F22, a protein of the cytochrome-P450 family 4, encodes an epidermal ω-hydroxylase decisive in the formation of acylceramides, which is hypothesized to be crucial for skin-barrier function. We report a girl with consanguineous parents presenting as collodion baby with contractures of the great joints and palmoplantar hyperlinearity. In the course of the disease she developed fine scaling of the skin with erythroderma, the latter disappearing until the age of 6 months. Her sister showed a generalized fine-scaling phenotype, and, interestingly, was born without a collodion membrane. The analysis of all known candidate genes for ARCI in parallel with a next-generation sequencing approach using a newly designed dermatogenetics gene panel revealed a previously unknown homozygous splice-site mutation c.549+5G>C in CYP4F22 in both girls, confirming the diagnosis of ARCI. Ultrastructural analysis by transmission electron microscopy in both patients showed epidermal hyperplasia, orthohyperkeratosis with persistence of corneodesmosomes into the outer stratum corneum layers, fragmented and disorganized lamellar lipid bilayers, which could be ascribed to inhomogeneous lamellar body secretion, as well as lamellar body and lipid entombment in the corneocytes. These findings correlated with increased transepidermal water loss on the functional level. For the first time, we report a collodion baby phenotype and epidermal barrier impairment in CyP4F22-deficient epidermis at both the ultrastructural and functional level, and corroborate the importance of CyP4F22 for epidermal maturation and barrier function.

Combining genomewide association study and lung eQTL analysis provides evidence for novel genes associated with asthma.

BACKGROUND: Genomewide association studies (GWASs) of asthma have identified single-nucleotide polymorphisms (SNPs) that modestly increase the risk for asthma. This could be due to phenotypic heterogeneity of asthma. Bronchial hyperresponsiveness (BHR) is a phenotypic hallmark of asthma. We aim to identify susceptibility genes for asthma combined with BHR and analyse the presence of cis-eQTLs among replicated SNPs. Secondly, we compare the genetic association of SNPs previously associated with (doctor's diagnosed) asthma to our GWAS of asthma with BHR. METHODS: A GWAS was performed in 920 asthmatics with BHR and 980 controls. Top SNPs of our GWAS were analysed in four replication cohorts, and lung cis-eQTL analysis was performed on replicated SNPs. We investigated association of SNPs previously associated with asthma in our data. RESULTS: A total of 368 SNPs were followed up for replication. Six SNPs in genes encoding ABI3BP, NAF1, MICA and the 17q21 locus replicated in one or more cohorts, with one locus (17q21) achieving genomewide significance after meta-analysis. Five of 6 replicated SNPs regulated 35 gene transcripts in whole lung. Eight of 20 asthma-associated SNPs from previous GWAS were significantly associated with asthma and BHR. Three SNPs, in IL-33 and GSDMB, showed larger effect sizes in our data compared to published literature. CONCLUSIONS: Combining GWAS with subsequent lung eQTL analysis revealed disease-associated SNPs regulating lung mRNA expression levels of potential new asthma genes. Adding BHR to the asthma definition does not lead to an overall larger genetic effect size than analysing (doctor's diagnosed) asthma.

The importance of genetic susceptibility in Dupuytren's disease.

Dupuytren's disease (DD) is a progressive fibromatosis that causes the formation of nodules and cords in the palmar aponeurosis leading to flexion contracture of affected fingers. The etiopathogenesis is multifactorial with a strong genetic predisposition. It is the most frequent genetic disorder of connective tissues. We have collected clinical data from 736 unrelated individuals with DD who underwent surgical treatment from Germany and Switzerland. We evaluated a standardised questionnaire, assessed the importance of different risk factors and compared subgroups with and without positive family history. We found that family history clearly had the strongest influence on the age at first surgery compared to environmental factors, followed by male sex. Participants with a positive family history were on average 55.9 years of age at the first surgical intervention, 5.2 years younger than probands without known family history (p = 6.7 × 10(-8) ). The percentage of familial cases decreased with age of onset from 55% in the 40-49 years old to 17% at age 80 years or older. Further risk factors analysed were cigarettes, alcohol, diabetes, hypertension, and epilepsy. Our data pinpoint the importance of genetic susceptibility for DD, which has long been underestimated.

Clinical and genetic findings in a family with NMNAT1-associated Leber congenital amaurosis: case report and review of the literature.

BACKGROUND: Leber congenital amaurosis (LCA) is a severe retinal dystrophy, typically manifesting in the first year of life. Mutations in more than 18 genes have been reported to date. In recent studies, biallelic mutations in NMNAT1 encoding nicotinamide mononucleotide adenylyltransferase 1 have been found to cause LCA. PURPOSE: To broaden the knowledge regarding the phenotype of NMNAT1-associated LCA. METHODS: Clinical ophthalmologic examinations were performed in two sisters with LCA. Whole exome sequencing was performed in one of the affected girls, with subsequent segregation analysis in the affected sister and unaffected parents. The literature was reviewed for reports of NMNAT1-associated LCA. RESULTS: Exome sequencing revealed the known NMNAT1 mutation c.25G>A (p.Val9Met) in a homozygous state. Segregation analysis showed the same homozygous mutation in the affected younger sister. Both parents were found to be heterozygous carriers of the mutation. The two girls both presented with severe visual impairment, nystagmus, central atrophy of the pigment epithelium, and pigment clumping in the periphery before the age of 6 months. Retinal vessels were attenuated. Both children were hyperopic. In the older sister, differential diagnosis included an inflammatory origin, but electrophysiology in her as well as her sister confirmed a diagnosis of LCA. Pallor of the optic nerve head was not present at birth but developed progressively. CONCLUSIONS: We confirmed a diagnosis of NMNAT1-associated LCA in two siblings through identification of the mutation (c.25G>A [p. Val9Met]) in a homozygous state. In infants with non-detectable electroretinogram (ERG), along with severe congenital visual dysfunction or blindness and central pigment epithelium atrophy with pigment clumping resembling scarring due to chorioretinitis, LCA due to NMNAT1 mutations should be considered.

Genetic heterogeneity in Pakistani microcephaly families.

Autosomal recessive primary microcephaly (MCPH) is caused by mutations in at least eight different genes involved either in cell division or DNA repair. Most mutations are identified in consanguine families from Pakistan, Iran and India. To further assess their genetic heterogeneity and mutational spectra, we have analyzed 57 consanguine Pakistani MCPH families. In 34 MCPH families, we detected linkage to five out of the eight well-characterized disease loci and identified mutations in 27 families, leaving seven families without mutations in the coding exons of the presumably underlying MCPH genes. In the MCPH cohort 23 families could not be linked to any of the known loci, pointing to remarkable locus heterogeneity. The majority of mutations were found in ASPM followed by WDR62, CENPJ, CEP152 and MCPH1. One ASPM mutation (p.Trp1326*) was found in as many as eight families suggesting a Pakistani founder mutation. One third of the families were linked to ASPM followed by WDR62 confirming previous data. We identified three novel ASPM mutations, four novel WDR62 mutations, one novel MCPH1 mutation and two novel CEP152 mutations. CEP152 mutations have not been described before in the Pakistani population.

Complete characterization of the edited transcriptome of the mitochondrion of Physarum polycephalum using deep sequencing of RNA.

RNAs transcribed from the mitochondrial genome of Physarum polycephalum are heavily edited. The most prevalent editing event is the insertion of single Cs, with Us and dinucleotides also added at specific sites. The existence of insertional editing makes gene identification difficult and localization of editing sites has relied upon characterization of individual cDNAs. We have now determined the complete mitochondrial transcriptome of Physarum using Illumina deep sequencing of purified mitochondrial RNA. We report the first instances of A and G insertions and sites of partial and extragenic editing in Physarum mitochondrial RNAs, as well as an additional 772 C, U and dinucleotide insertions. The notable lack of antisense RNAs in our non-size selected, directional library argues strongly against an RNA-guided editing mechanism. Also of interest are our findings that sites of C to U changes are unedited at a significantly higher frequency than insertional editing sites and that substitutional editing of neighboring sites appears to be coupled. Finally, in addition to the characterization of RNAs from 17 predicted genes, our data identified nine new mitochondrial genes, four of which encode proteins that do not resemble other proteins in the database. Curiously, one of the latter mRNAs contains no editing sites.

Heterozygous mutations in ANKH, the human ortholog of the mouse progressive ankylosis gene, result in craniometaphyseal dysplasia.

Craniometaphyseal dysplasia (CMD) is a bone dysplasia characterized by overgrowth and sclerosis of the craniofacial bones and abnormal modeling of the metaphyses of the tubular bones. Hyperostosis and sclerosis of the skull may lead to cranial nerve compressions resulting in hearing loss and facial palsy. An autosomal dominant form of the disorder (MIM 123000) was linked to chromosome 5p15.2-p14.1 (ref. 3) within a region harboring the human homolog (ANKH) of the mouse progressive ankylosis (ank) gene. The ANK protein spans the outer cell membrane and shuttles inorganic pyrophosphate (PPi), a major inhibitor of physiologic and pathologic calcification, bone mineralization and bone resorption. Here we carry out mutation analysis of ANKH, revealing six different mutations in eight of nine families. The mutations predict single amino acid substitutions, deletions or insertions. Using a helix prediction program, we propose for the ANK molecule 12 membrane-spanning helices with an alternate inside/out orientation and a central channel permitting the passage of PPi. The mutations occur at highly conserved amino acid residues presumed to be located in the cytosolic portion of the protein. Our results link the PPi channel ANK with bone formation and remodeling.

Mutations in the skeletal muscle alpha-actin gene in patients with actin myopathy and nemaline myopathy.

Muscle contraction results from the force generated between the thin filament protein actin and the thick filament protein myosin, which causes the thick and thin muscle filaments to slide past each other. There are skeletal muscle, cardiac muscle, smooth muscle and non-muscle isoforms of both actin and myosin. Inherited diseases in humans have been associated with defects in cardiac actin (dilated cardiomyopathy and hypertrophic cardiomyopathy), cardiac myosin (hypertrophic cardiomyopathy) and non-muscle myosin (deafness). Here we report that mutations in the human skeletal muscle alpha-actin gene (ACTA1) are associated with two different muscle diseases, 'congenital myopathy with excess of thin myofilaments' (actin myopathy) and nemaline myopathy. Both diseases are characterized by structural abnormalities of the muscle fibres and variable degrees of muscle weakness. We have identified 15 different missense mutations resulting in 14 different amino acid changes. The missense mutations in ACTA1 are distributed throughout all six coding exons, and some involve known functional domains of actin. Approximately half of the patients died within their first year, but two female patients have survived into their thirties and have children. We identified dominant mutations in all but 1 of 14 families, with the missense mutations being single and heterozygous. The only family showing dominant inheritance comprised a 33-year-old affected mother and her two affected and two unaffected children. In another family, the clinically unaffected father is a somatic mosaic for the mutation seen in both of his affected children. We identified recessive mutations in one family in which the two affected siblings had heterozygous mutations in two different exons, one paternally and the other maternally inherited. We also identified de novo mutations in seven sporadic probands for which it was possible to analyse parental DNA.

Congenital disorder of glycosylation type Ij (CDG-Ij, DPAGT1-CDG): extending the clinical and molecular spectrum of a rare disease.

Congenital disorders of glycosylation (CDG) are caused by enzymatic defects of the formation or processing of lipid-linked oligosaccharides and glycoproteins. Since the majority of proteins is glycosylated, a defect in a singular CDG enzyme leads to a multisytemic disease with secondary malfunction of thousands of proteins. CDG-Ij (DPAGT1-CDG) is caused by a defect of the human DPAGT1 (UDP-GlcNAc: Dolichol Phosphate N-Acetylglucosamine-1-Phosphotransferase), catalyzing the first step of N-linked glycosylation. So far the clinical phenotype of only one CDG-Ij patient has been described. The patient showed severe muscular hypotonia, intractable seizures, developmental delay, mental retardation, microcephaly and exotropia. Molecular studies of this patient revealed the heterozygous mutation c.660A>G (Y170C; paternal) in combination with an uncharacterized splicing defect (maternal). Two further mutations, c.890A>T (I297F) and c.162-8G>A as a splicing defect were detected when analyzing DPAGT1 in two affected siblings of a second family. We report two new patients with the novel homozygous mutation, c.341C>G (A114 G), causing a severe clinical phenotype, characterized by hyperexcitability, intractable seizures, bilateral cataracts, progressive microcephaly and muscular hypotonia. Both our patients died within their first year of life. With the discovery of this novel mutation and a detailed clinical description we extend the clinical features of CDG-Ij in order to improve early detection of this disease.

First HPSE2 missense mutation in urofacial syndrome.

Urofacial syndrome (UFS) describes the combination of urological problems and an inverted facial expression upon attempts to smile. Seventeen independent familial cases from different ethnicities have been described so far. Some of these have been linked to chromosome 10q. Very recently, homozygous loss-of-function mutations affecting the gene HPSE2 were identified in nine cases. Here, we describe a consanguineous UFS family from Pakistan with three of six siblings affected. We establish linkage to the chromosome 10q critical region and identify two non-synonymous HPSE2 variants. In silico analysis and screening of controls defines c.631T>C (p.Y211H) as a novel benign SNP and c.1628A>T (p.N543I) as the disease-causing mutation. Our study exemplifies the challenges in proper clinical diagnosis of UFS and, thereby, supports the hypothesis of the disease being under diagnosed. By identifying the first HPSE2 missense mutation it also provides a starting point for studies aimed at functionally understanding the unusual combination of symptoms as characterizing UFS.

Phenotypic variability in a large Czech family with a dynamin 2-associated Charcot-Marie-Tooth neuropathy.

Mutations in the Dynamin 2 gene (DNM2) cause autosomal dominant centronuclear myopathy or autosomal dominant (AD) Charcot-Marie-Tooth (CMT) disease. Here the authors report one large Czech family with 15 members affected with an AD CMT phenotype of extraordinary variability. Genetic linkage analysis using SNP arrays revealed a locus of about 9.6 Mb on chromosome 19p13.1-13.2. In this critical interval, 373 genes were located. The only gene herein known to be associated with an intermediate type of CMT was Dynamin 2 (DNM2). Subsequent sequence analysis of the DNM2 gene in the index patient revealed a novel missense mutation p.Met580Thr. This missense mutation segregated with the neuropathy, indicating the causal character of this mutation. The phenotype of CMT in this family shows mild to moderate impairment with relatively preserved upper limbs and a very broad range of the onset of clinical symptoms from an early onset around the age of 12 to the late onset during the fifth decade. Electrophysiology showed an intermediate type of peripheral neuropathy. The motor median nerve conduction velocity varied from 36 m/s to normal values with signs of asymmetrical affection of peripheral nerves. No additional symptoms such as cranial nerve involvement, cataract, and signs of neutropenia or myopathy syndrome were observed in any member of the family yet. The progression was slow with no loss of ambulation. The authors suggest that the characterization of clinical variability in a single family may help to direct the genetic analysis directly to the rarely observed DNM2 mutations.

Hypomorphic mutations in meckelin (MKS3/TMEM67) cause nephronophthisis with liver fibrosis (NPHP11).

BACKGROUND: Nephronophthisis (NPHP), a rare recessive cystic kidney disease, is the most frequent genetic cause of chronic renal failure in children and young adults. Mutations in nine genes (NPHP1-9) have been identified. NPHP can be associated with retinal degeneration (Senior-Løken syndrome), brainstem and cerebellar anomalies (Joubert syndrome), or liver fibrosis. METHODS: To identify a causative gene for the subset of patients with associated liver fibrosis, the authors performed a genome wide linkage search in a consanguineous family with three affected patients using 50K SNP microarrays and homozygosity mapping. RESULTS: The authors obtained a significant maximum parametric LOD (logarithm of odds) score of Z(max) = 3.72 on chromosome 8q22 and identified a homozygous missense mutation in the gene MKS3/TMEM67. When examining a worldwide cohort of 62 independent patients with NPHP and associated liver fibrosis we identified altogether four novel mutations (p.W290L, p.C615R, p.G821S, and p.G821R) in five of them. Mutations of MKS3/TMEM67, found recently in Meckel-Gruber syndrome (MKS) type 3 and Joubert syndrome (JBTS) type 6, are predominantly truncating mutations. In contrast, the mutations detected here in patients with NPHP and associated liver fibrosis are exclusively missense mutations. This suggests that they may represent hypomorphic alleles, leading to a milder phenotype compared with the more severe MKS or JBTS phenotype. Additionally, mutation analysis for MKS3/TMEM67 in 120 patients with JBTS yielded seven different (four novel) mutations in five patients, four of whom also presented with congenital liver fibrosis. CONCLUSIONS: Hypomorphic MKS3/TMEM67 mutations cause NPHP with liver fibrosis (NPHP11). This is the first report of MKS3 mutations in patients with no vermian agenesis and without neurological signs. Thus NPHP, JBTS, and MKS represent allelic disorders.

Mhc class I haplotypes associated with survival time in simian immunodeficiency virus (SIV)-infected rhesus macaques.

In both human immunodeficiency virus-infected humans and simian immunodeficiency virus (SIV)-infected macaques, genes encoded in the major histocompatibility complex (MHC) class I region are important determinants of disease progression. However, compared to the human human lymphocyte antigen complex, the macaque MHC region encodes many more class I genes. Macaques with the same immunodominant class I genes express additional Mhc genes with the potential to influence the disease course. We therefore assessed the association between of the Mhc class I haplotypes, rather than single gene variants, and survival time in SIV-infected rhesus macaques (Macaca mulatta). DNA sequence analysis and Mhc genotyping of 245 pedigreed monkeys identified 17 Mhc class I haplotypes that constitute 10 major genotypes. Among 81 vaccination-naive, SIV-infected macaques, 71 monkeys carried at least one Mhc class I haplotype encoding only MHC antigens that were incapable of inducing an effective anti-SIV cytotoxic T lymphocytes response. Study of these macaques enabled us to relate individual Mhc class I haplotypes to slow, medium and rapid disease progression. In a post hoc analysis, classification according to disease progression was found to explain at least 48% of the observed variation of survival time.