19 F 58 B A VHL T653C I147T

A female patient was diagnosed with a 3cm, right adrenal, metanephrine-secreting (PHEO) at age 47, and 11 years later, developed a Furhman grade I-II RCC with typical features of clear cell type. She remains disease-free after 18 and 7 years of follow up, respectively. She had two siblings with PHEOs, detected at 44 and 51 years of age, but no other RCCs. A germline truncating TMEM127 mutation, c.532_533dupT; p.Y178LfsX48, hereafter referred to as TMEM127-532dupT, was identified after a targeted, exome-based next generation sequencing screening (Fig.1C). No other pathogenic germline mutation was detected in other 35 pheochromocytoma and/or renal cancer susceptibility genes screened. Furthermore, no somatic mutations were found at high-depth sequencing (average 180x) of the proband’s frozen PHEO using Illumina TruSeq Cancer Panel screening of 42 cancer genes. The TMEM127-532dupT mutation was also detected in germline DNA from one affected sister (Fig.1A, ​,1B)1B) and three other family members, their mother and two brothers, who have no evidence of PHEO or RCC (Fig. 1A). The proband’s mother was diagnosed with a lung adenocarcinoma at age 78. Analysis of the proband’s fresh-frozen PHEO DNA revealed loss of the wild-type TMEM127 allele (Fig.1B). These results support the germline TMEM127 mutation as the main driver event in this family (Qin et al. 2010). In contrast, no TMEM127 loss was found in three separate regions dissected from the proband’s formalin-fixed paraffin embedded (FFPE) RCC (Fig.1C). Similar to the proband’s PHEO, the FFPE PHEO from the proband’s sister displayed clear loss of heterozygosity (LOH) of the wild-type allele (Fig.1C), excluding potential artifacts due to fixed tissue quality. The lack of TMEM127 LOH in the RCC is in agreement with our earlier observation of retention of heterozygosity in a limited set of renal cancers with germline TMEM127 mutations(Qin et al. 2014). Interestingly, in this earlier report we noted decreased TMEM127 transcription in the tumors carrying these heterozygous variants, potentially suggesting a dosage effect of the TMEM127 mutation in renal tissue.Open in a separate windowFigure 1A) Partial pedigree of a family with a germline TMEM127 mutation (c.532dupT, p.Y178LfsX48). Clinical features are indicated in the labels. Clinically unaffected mutation carriers are 87 (I-2), 62 (II-3) and 60 (II-4) years old. One additional, 54-year-old sibling (not shown) is negative for the mutation. B) Electropherograms of germline DNA from the proband (II-1) and one affected sister (II-2), and fresh-frozen PHEO from the proband, compared to the reference sequence. The single nucleotide insertion site is indicated with an arrow; the PHEO sample has no wild-type allele sequence. C) Electropherograms of archival, formalin-fixed and paraffin embedded tumor DNA: PHEO from affected sister and three separate subregions of the proband’s RCC, referred to as RCC-1, RCC-2 and RCC-3, compared to the reference sequence. Note loss of the wild-type allele in the PHEO but not in the RCC subregions. D) Western blot of lysates from the proband’s PHEO, two unrelated PHEOs carrying distinct TMEM127 mutations, and four PHEOS of other genetic backgrounds (mutated gene indicated), a separate gel of HEK293T cells expressing wild-type or c.532dupT TMEM127 construct containing an HA tag are shown to indicate the expected size of truncated c.532dupT TMEM127; Membranes were probed with TMEM127 (Bethyl Labs), phospho-Thr389- S6 kinase (S6K) and total S6K (Cell Signaling Technology), and β-actin (Sigma) as a loading control. E) Confocal microscopy images of HEK293T cells transiently transfected with GFP-tagged wild-type (WT) TMEM127 and GFP-TMEM127-532dupT mutant protein; a previously reported pathogenic mutant, GFP-TMEM127-S147del, was included as a positive control. WT-TMEM127 has a punctate pattern, S147del is diffusely distributed, and 532dupT is predominantly diffuse. F) GuideRNA (sgRNA) sequence used to genome-edit HEK293 cells, indicating the protospacer adjacent motif (PAM) sequence and the position of the nucleotide insertion of a clone. Corresponding electropherogram is shown below (arrow indicates inserted T at position c.532 on the TMEM127 sequence).G) Western blot of lysates from HEK293 control and 532dupT cells after amino acid starvation followed by exposure for 15 minutes. No TMEM127 protein is detected in the HEK293-TMEM127-532dupT cells. Phosphorylation of mTOR targets S6 kinase (S6K) and downstream S6 are shown and β-actin was used as a loading control. H) Electropherograms of germline and tumor samples spanning a VHL 3’UTR SNP (dbSNP rs1642742) used to estimate copy number at the VHL gene. DNA from proband’s germline and archival RCCs, as well as PHEO from proband and her sister PHEO. Percentage of the ‘A’ allele representation of each sample was estimated by the Mutation Surveyor program, as reported(Toledo, et al. 2016).To further investigate whether the TMEM127-532dupT mutation was pathogenic, we explored its consequences in tumor tissue. No frozen material was available from the RCC and, in the absence of a reliable TMEM127 antibody for immunohistochemistry, no additional analysis of TMEM127 protein was available from this tumor. However, Western blot of protein lysates from the proband’s frozen PHEO using a polyclonal antibody that recognizes the TMEM127 N-terminus (Bethyl labs) (Qin et al. 2014) revealed neither full-length nor truncated TMEM127 bands (Fig 1D). Other TMEM127-mutant PHEOS included as controls also showed no TMEM127 expression, while TMEM127 protein was clearly detectable in PHEOs of other genetic origins but with intact TMEM127 sequence (Fig.1D), in support of instability of mutant TMEM127 protein. In addition, in agreement with our previous observations suggesting that the mTORC1 kinase pathway is activated after TMEM127 loss (Qin et al. 2010), phosphorylation of the mTORC1 downstream target S6 kinase (S6K) was increased in the TMEM127-532dupT PHEO and in the other TMEM127 mutant tumors, compared to TMEM127 wild-type PHEOs (Fig.1D).Next, we examined TMEM127 subcellular distribution as another functional readout by expressing in HEK293T kidney cells a construct carrying this mutation fused with a green fluorescent protein (GFP) made by site directed mutagenesis, as described (Qin et al. 2010). The TMEM127-532dupT mutant showed a diffuse cytoplasmic pattern, similar to a previously reported pathogenic TMEM127 mutant (Qin et al. 2014), but distinct from the punctate endomembrane distribution of wild type TMEM127 (Fig.1E). We were unable to generate cells that retained stable expression of this mutant, further supporting its instability. To study the effects of the mutation in a more physiological context in renal cells, we generated HEK293 cells carrying homozygous TMEM127-532dupT mutation by CRISPR-Cas9-based genome modification using previously published protocols(Sanjana, et al. 2014) and a guide RNA that targeted the mutated nucleotide site or a control (Fig.1F). Stable clones carrying the c.532dupT mutation in homozygosity were obtained and verified by sequencing (Fig 1F). Similar to the proband’s PHEO, HEK293 TMEM127-532dupT cells had no detectable TMEM127 protein (Fig1G). Moreover, incubation of these cells with amino acids following 2 hours of amino acid deprivation, a powerful mTORC1 pathway activation input(Sancak, et al. 2010), led to higher mTOR target phosphorylation in HEK293 TMEM127-532dupT cells compared with control (Fig. 1G), suggesting that kidney cells with mutant TMEM127-532dupT have increased mTORC1 activation. Taken together, our data indicate that the TMEM127-532dupT mutation leads to loss of TMEM127 function both in primary PHEO and in renal cells, consistent with its pathogenic role.In view of the lack of TMEM127 LOH in the RCC, we investigated other possible genetic causes. Immunohistochemistry for SDHB was positive, excluding an SDH mutation. We sequenced the coding region of the VHL gene in three subsections of proband’s RCC and found no mutations. We also evaluated VHL copy number using a SNP located in the 3’UTR of the gene (dbSNP rs1642742, c.*294G>A) that was heterozygous in the proband germline

F58blueFemale58delC, fsThr1051st R (1 prior L plus L total)No22.65Yes

geopolitics

RRID:ZFIN_ZDB-GENO-110131-58

Results for individual PALB2 variants were normalized relative to WT-PALB2 and the p.Tyr551ter (p.Y551X) truncating variant on a 1:5 scale with the fold change in GFP-positive cells for WT set at 5.0 and fold change GFP-positive cells for p.Y551X set at 1.0. The p.L24S (c.71T>C), p.L35P (c.104T>C), p.I944N (c.2831T>A), and p.L1070P (c.3209T>C) variants and all protein-truncating frame-shift and deletion variants tested were deficient in HDR activity, with normalized fold change <2.0 (approximately 40% activity) (Fig. 1a).

A total of 84 PALB2 patient-derived missense variants reported in ClinVar, COSMIC, and the PALB2 LOVD database were selected

Source Data

Source Data

We, therefore, analyzed the effect of 48 PALB2 VUS (Fig. 2a, blue) and one synthetic missense variant (p.A1025R) (Fig. 2a, purple)29 on PALB2 function in HR.

Most Suspected Brugada Syndrome Variants Had (Partial) Loss of Function

we selected 73 previously unstudied variants: 63 suspected Brugada syndrome variants and 10 suspected benign variants

ZFIN_ZDB-GENO-110131-58

Again, the establishment of a Federal or Federative union would, as l believe, be immediately followed by an agitation in favour of the election or the Local Governors, instead of their being nominated by the Crown. And it would be the more difficult to resist this application on account of the purely local or municipal character of the powers with which the Governors would he intrusted ; but the compliance with the request would he in my opinion-highly dangerous, not only because it would at once be fatal to British influence in the Local Governments and Local Legislatures, but also because it would, I believe, be followed quickly by a similar application from the United ‘Provinces, with regard to the Gov,ernor-Generalship, still more difficult to resist from the force With which it would be pressed, but the compliance with which would at once practically sever the connexion between the Crown and British North America.

The honorable member has taken occasion to find fault with the clause of the resolutions which provides that the lieutenant-governors shall be appointed by the Central Government, and sees in it great danger, especially to Lower Canada. Mr. SPEAKER, I should very much like to know what protection the population of the different provinces derive from the fact that the governors of the British North American Provinces are sent out to us from England. Under the existing system, our governor is responsible neither to the people nor to the House ; he depends entirely upon the English Government, to which he is responsible. Under the system proposed the lieutenant-governors will be appointed by the Central Government, to which they will necessarily be responsible for their actions. And in that Government we shall have more than one vote ; we shall be represented in it by our ministers, who will be there to cause every encroachment or arbitrary act which the lieutenant-governor may allow himself to commit, to be condemned.

With respect to the local governments, it is provided that each shall be governed by a chief executive officer, who shall be nominated by the General Government. As this is to be one united province, with the local governments and legislatures subordinate to the General Government and Legislature, it is obvious that the chief executive officer in each of the provinces must be subordinate as well. The General Government assumes towards the local governments precisely the same position as the Imperial Government holds with respect to each of the colonies now; so that as the Lieutenant Governor of each of the different provinces is now appointed directly by the Queen, and is directly responsible, and reports directly to Her, so will the executives of the local governments hereafter be subordinate to the Representative of the Queen, and be responsible and report to him. Objection has been taken that there is an infringement of the Royal prerogative in giving the pardoning power to the local governors, who are not appointed directly by the Crown, but only indirectly by the Chief Executive of the Confederation, who is appointed by the Crown. This provision was inserted in the Constitution on account of the practical difficulty which must arise if the power is confined to the Governor General. For example, if a question arose about the discharge of a prisoner convicted of a minor offence, say in Newfoundland, who might be in imminent danger of losing his life if he remained in confinement, the exercise of the pardoning power might come too late if it were necessary to wait for the action of the Governor General. It must be remembered that the pardoning power not only extends to capital cases, but to every case of conviction and sentence, no matter how trifling— even to the case of a fine in the nature of a sentence on a criminal conviction. It extends to innumerable cases, where, if the responsibility for its exercise were thrown on the General Executive, it could not be so satisfactorily discharged, Of course there must be, in each province, a legal adviser of the Executive, occupying the position of our Attorney General, as there is in every state of the American Union. This officer will be an officer of the Local Government ; but, if the pardoning power is reserved for the Chief Executive, there must, in every case where the exercise of the pardoning power is sought, be a direct communication and report from the local law officer to the Governor General. The practical inconvenience of this was felt to be so great, that it was thought well to propose the arrangement we did, without any desire to infringe upon the prerogatives of the Crown, for our whole action shows that the Conference, in every step they took, were actuated by a desire to guard jealously these prerogatives.

With regard to the acts of the local legislatures, it was proposed they should, in like manner, either be reserved by the Lieutenant Governor, or should, if assented to by him, be liable to disallowance by the general government within one year.

It was felt that more advantage would arise by making the reference from the local to the general legislature direct than to have it go through the Colonial Office. It was plain the Queen, or Sovereign authority, must have the right of exercising the power of controlling legislation in the way which had existed for so many years in every part of the British dominions. There would be no object in sending over mere local bills to the Colonial Office or to the Queen for sanction. It was felt that points on which differences might arise on local bills would be better understood by ourselves in this country than by the Imperial authorities. If reserved, they would have to be referred back to the General Government for its advice as to their disposal ; and if this advice were given, the parties concerned would be ignorant of the advisers, who could not be held responsible. The principle upon which our Government was administered was, that no act was done without some one being responsible. It was desirable therefore that such advice should be tendered by parties who could be brought to account for it by the representatives from the section of the country concerned, in the General Parliament.

For, while they would be selected from among ourselves, they would be required to administer the Governments of their respective Provinces, not according to their own will and pleasure but according to the advice of officers who possessed the confidence of the Local Legislatures of those Provinces. Consequently we should always have the means of bringing about harmony, if any difficulty arose between any of the local bodies and the General Government, through the Lieutenant Governor, and we should have a system under which, all action beginning with the people and proceeding through the Local Legislature, would, before it became law, come under the revision of the Lieutenant Governor, who would be responsible for his action, and be obliged to made his report to the superior authority.

It was well that there should be those objects of ambition. At present the Bar and the Bench might be said to possess almost the only prizes the country offered to its public men. It was desirable, he thought, that we should have within our reach the opportunity of rewarding merit by appointing from among ourselves in the several Provinces those who should be the heads of the Local Governments and who should form the links of connection between the Local Governments and the General Government, holding to that General Government the same relations as were now held by the heads of the Provincial Governments to the imperial Government, and discharging the duties [Page 14] of their offices under the same local advice as that which the Governors now acted on.

it was felt there was no necessity whatever for their being in communication with the Imperial Government, but that on the contrary very great mischief might arise, if they were permitted to bold that communication. It was also thought that, in keeping the appointment of the Lieutenant Governors in the hands of the General Government, this further advantage would be gained ; the appointments would be conferred on men in our own country. (Hear, hear.) There would be a selection from the public men of intelligence and standing in the respective Provinces, and they would go to the discharge of the duties imposed upon them with experience gained in public life in the colonies whose local affairs they were called on to administer, so that they would carry to the administration of public affairs in the respective Provinces that valuable acquaintance with the feelings and habits of thought of the people which they had gained during their public life.

And the first change he had to draw their attention to was with reference to the appointment of the Lieutenant Governor who it was proposed should be appointed by the General Government. The reason why this was preferred to the appointment taking place as heretofore by the Crown was that it was intended that the communication between all the several Provinces and the Imperial Government should be restricted to the General Government.