Mutations in ERGIC1 cause Arthrogryposis multiplex congenita, neuropathic type.

Arthrogryposis multiplex congenita (AMC) is heterogeneous group of disorders characterized by non-progressive joint contractures from birth that involve more than 1 part of the body. There are various etiologies for AMC including genetic and environmental depends on the specific type, however, for most types, the cause is not fully understood. We previously reported large Israeli Arab kindred consisting of 16 patients affected with AMC neuropathic type, and mapped the locus to a 5.5 cM interval on chromosome 5qter. Using whole exome sequencing, we have now identified homozygous pathogenic variant in the ERGIC1 gene within the previously defined linked region. ERGIC1 encodes a cycling membrane protein which has a possible role in transport between endoplasmic reticulum and Golgi. We further show that this mutation was absent in more than 200 samples of healthy unrelated individuals of the Israeli Arab population. Thus, our findings expand the spectrum of hereditary AMC and suggest that abnormalities in protein trafficking may underlie AMC-related disorders.

Prenatal course of metaphyseal anadysplasia associated with homozygous mutation in MMP9 identified by exome sequencing.

Metaphyseal anadysplasia (MANDP) is a rare autosomal recessive form of skeletal dysplasia characterized by normal length at birth and transitory bowing of the legs. Although several families with MANDP have been reported, homozygous mutations in the matrix metalloproteinase type 9 (MMP9) gene have been described in only one consanguineous family, and thus the pre and postnatal phenotypic spectrum is still obscure. A clinically similar but more severe type is caused by autosomal-dominant inheritance and is caused by mutations in matrix metalloproteinase type 13 gene (MMP13). Here, we report the prenatal and early postnatal course of two affected sib fetuses with early sonographic evidence of long bone shortening and postnatally no metaphyseal changes. Whole-exome sequencing revealed homozygous mutation in MMP9 in both fetuses suggesting a diagnosis of MANDP. We propose that MANDP should be considered in pregnancies with early prenatal shortening of the long bones without associated finding of lethal skeletal dysplasias. In addition, the finding of homozygous mutation in non-consanguineous parents of Jewish-Caucasus ancestry may suggest unawareness of such relation or the occurrence of a founder mutation in this gene.

Chromosomal microarray analysis in fetuses with aberrant right subclavian artery.

OBJECTIVE: To evaluate the association between aberrant right subclavian artery (ARSA), with or without additional risk factors for aneuploidy or ultrasound abnormality, and results of chromosomal microarray analysis (CMA). METHODS: This was a multicenter study of fetuses diagnosed with ARSA that underwent genetic analysis by CMA, all samples being analyzed in the same laboratory. Clinical investigation included nuchal translucency measurement, first- and second-trimester maternal serum screening, early and late second-trimester fetal anatomy scans and fetal echocardiography. Comparative genomic hybridization microarray analysis or single-nucleotide polymorphism array technology was used for CMA of DNA samples obtained from amniotic fluid. RESULTS: CMA results were available for 63 fetuses with ARSA. In 36 fetuses, ARSA was an isolated finding, and no pathogenic variant was found. Additional ultrasound findings and/or risk factors for aneuploidy were present in 27 fetuses, five of which had pathogenic CMA results. Of these five, trisomy 21 was detected in a fetus with echogenic intracardiac focus (EIF), 22q11 deletion was detected in a fetus with EIF and an increased risk of trisomy 21 of 1:230 from maternal serum screening, 22q11 duplication was detected in a fetus with hypoplastic right kidney and choroid plexus cyst and 22q11 deletion was detected in a fetus with right aortic arch and clubfoot. The fifth fetus had increased nuchal translucency thickness (4 mm) and a ventricular septal defect, and CMA identified both 22q11 deletion and 1q21 duplication. CONCLUSIONS: In fetuses with isolated ARSA, an invasive procedure for CMA is not indicated. However, CMA is recommended when additional ultrasound abnormalities or risk factors for aneuploidy are observed. The chromosomal findings in four of the five cases with an abnormal CMA result in our study would not have been detected by standard fetal chromosomal testing. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.

Ehlers-Danlos arthrochalasia type (VIIA-B)--expanding the phenotype: from prenatal life through adulthood.

The Ehlers-Danlos syndromes (EDS) form a clinically and genetically heterogeneous group of inherited connective-tissue disorders characterized by joint hypermobility, tissue fragility and skin abnormalities. Six subtypes have been well characterized based on clinical features and molecular genetic abnormalities. The arthrochalasia type EDS (formerly types VIIA and B) is characterized by severe generalized joint hypermobility with multiple dislocations including congenital bilateral dislocation of the hips, muscular hypotonia and distinct dysmorphic features. The diagnosis of the arthrochalasia type EDS is of importance in the neonatal period because of consequences of physical disability in later life. However, the differential diagnosis may be difficult because of overlap with other hypermobility syndromes. In addition, the significant hypotonia may direct the physician toward various neuromuscular diagnoses. As patients become older, the hypotonia decreases and facial features become less distinct. In this report, we describe seven patients at different ages. Timing of diagnosis varied from prenatal life to adult age. The diagnosis of EDS type VII was confirmed by biochemical studies or mutation analysis showing characteristic mutations in COL1A1 and COL1A2. These mutations result in skipping of exon 6, which leads to defective collagen synthesis. For physicians treating patients with EDS type VII, achieving mobility for the patient is the greatest challenge and it may be impossible because of recurrent dislocations of nearly all joints in severe cases.

The nuclear ubiquitin-proteasome system degrades MyoD.

Many short-lived nuclear proteins are targeted for degradation by the ubiquitin-proteasome pathway. The role of the nucleus in regulating the turnover of these proteins is not well defined, although many components of the ubiquitin-proteasome system are localized in the nucleus. We have used nucleoplasm from highly purified HeLa nuclei to examine the degradation of a physiological substrate of the ubiquitin-proteasome system (MyoD). In vitro studies using inhibitors of the system demonstrate MyoD is degraded via the ubiquitin-proteasome pathway in HeLa nucleoplasm. Purified nucleoplasm in vitro also supports the generation of high molecular mass MyoD-ubiquitin adducts. In addition, in vivo studies, using leptomycin B to inhibit nuclear export, demonstrate that MyoD is degraded in HeLa cells by the nuclear ubiquitin-proteasome system.

Degradation of the E7 human papillomavirus oncoprotein by the ubiquitin-proteasome system: targeting via ubiquitination of the N-terminal residue.

The E7 oncoprotein of the high risk human papillomavirus type 16 (HPV-16), which is etiologically associated with uterine cervical cancer, is a potent immortalizing and transforming agent. It probably exerts its oncogenic functions by interacting and altering the normal activity of cell cycle control proteins such as p21WAF1, p27KIP1 and pRb, transcriptional activators such as TBP and AP-1, and metabolic regulators such as M2-pyruvate kinase (M2-PK). Here we show that E7 is a short-lived protein and its degradation both in vitro and in vivo is mediated by the ubiquitin-proteasome pathway. Interestingly, ubiquitin does not attach to any of the two internal Lysine residues of E7. Substitution of these residues with Arg does not affect the ability of the protein to be conjugated and degraded; in contrast, addition of a Myc tag to the N-terminal but not to the C-terminal residue, stabilizes the protein. Also, deletion of the first 11 amino acid residues stabilizes the protein in cells. Taken together, these findings strongly suggest that, like MyoD and the Epstein Barr Virus (EBV) transforming Latent Membrane Protein 1 (LMPI), the first ubiquitin moiety is attached linearly to the free N-terminal residue of E7. Additional ubiquitin moieties are then attached to an internal Lys residue of the previously conjugated molecule. The involvement of E7 in many diverse and apparently unrelated processes requires tight regulation of its function and cellular level, which is controlled in this case by ubiquitin-mediated proteolysis.

Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice.

Mutant ataxin-1, the expanded polyglutamine protein causing spinocerebellar ataxia type 1 (SCA1), aggregates in ubiquitin-positive nuclear inclusions (NI) that alter proteasome distribution in affected SCA1 patient neurons. Here, we observed that ataxin-1 is degraded by the ubiquitin-proteasome pathway. While ataxin-1 [2Q] and mutant ataxin-1 [92Q] are polyubiquitinated equally well in vitro, the mutant form is three times more resistant to degradation. Inhibiting proteasomal degradation promotes ataxin-1 aggregation in transfected cells. And in mice, Purkinje cells that express mutant ataxin-1 but not a ubiquitin-protein ligase have significantly fewer NIs. Nonetheless, the Purkinje cell pathology is markedly worse than that of SCA1 mice. Taken together, NIs are not necessary to induce neurodegeneration, but impaired proteasomal degradation of mutant ataxin-1 may contribute to SCA1 pathogenesis.

Basal and human papillomavirus E6 oncoprotein-induced degradation of Myc proteins by the ubiquitin pathway.

We have previously shown that the degradation of c-myc and N-myc in vitro is mediated by the ubiquitin system. However, the role of the system in targeting the myc proteins in vivo and the identity of the conjugating enzymes and possible ancillary proteins involved has remained obscure. Here we report that the degradation of the myc proteins in cells is inhibited by lactacystin and MG132, two inhibitors of the 20S proteasome. Inhibition is accompanied by accumulation of myc-ubiquitin conjugates. Dissection of the ancillary proteins involved revealed that the high-risk human papillomavirus oncoprotein E6-16 stimulates conjugation and subsequent degradation of the myc proteins in vitro. Expression of E6-16 in cells results in significant shortening of the t1/2 of the myc proteins with subsequent decrease in their cellular level. Analysis of the conjugating enzymes revealed that under basal conditions the proteins can be conjugated by two pairs of E2s and E3s-E2-14 kDa and E3alpha involved in the "N-end rule" pathway, and E2-F1 (UbcH7) and E3-Fos involved also in conjugation of c-Fos. In the presence of E6-16, a third pair, E2-F1 and E6-AP mediate conjugation of myc by means of a mechanism that appears to be similar to that involved in the targeting of p53, formation of a myc. E6.E6-AP targeting complex. It is possible that in certain cells E6-mediated targeting of myc prevents myc-induced apoptosis and thus ensures maintenance of viral infection.