A systematic strategy was used to create a synoptic set of mutations that are distributed throughout the single 0-tubulin gene of Saccharomyces cerevisiae. Clusters of charged amino acids were targeted for mutagenesis and converted to alanine to maximize alterations on the protein's surface and minimize alterations that affect protein folding. Of the 55 mutations we constructed, three confer dominant-lethality, 11 confer recessive-lethality, 10 confer cold-sensitivity, one confers heat-sensitivity, and 27 confer altered resistance to benomyl. Only 11 alleles give no discernible phenotype. In spite of the fact that f-tubulin is a highly conserved protein, three-fourths of the mutations do not destroy the ability of the protein to support the growth of yeast at 30°C. The lethal substitutions are primarily located in three regions of the protein and presumably identify domains most critical for 0-tubulin function. Interestingly, most of the conditional-lethal alleles produce specific defects in spindle assembly at their restrictive temperature; cytoplasmic microtubules are relatively unaffected. The exceptions are two mutants that contain abnormally long cytoplasmic microtubules. Mutants with specific spindle defects were not observed in our previous collection of f-tubulin mutants and should be valuable in dissecting spindle function.

Charcot-Marie-Tooth type 4 (CMT4) is an autosomal recessive severe form of neuropathy with genetic heterogeneity. CMT4B1 is caused by mutations in the myotubularin-related 2 (MTMR2) gene and as a member of the myotubularin family, the MTMR2 protein is crucial for the modulation of membrane trafficking. To enable future clinical trials, we performed a detailed review of the published cases with MTMR2 mutations and describe four novel cases identified through whole-exome sequencing (WES). The four unrelated families harbor novel homozygous mutations in MTMR2 (NM_016156, Family 1: c.1490dupC; p.Phe498IlefsTer2; Family 2: c.1479+1G>A; Family 3: c.1090C>T; p.Arg364Ter; Family 4: c.883C>T; p.Arg295Ter) and present with CMT4B1-related severe early-onset motor and sensory neuropathy, generalized muscle atrophy, facial and bulbar weakness, and pes cavus deformity. The clinical description of the new mutations reported here overlap with previously reported CMT4B1 phenotypes caused by mutations in the phosphatase domain of MTMR2, suggesting that nonsense MTMR2 mutations, which are predicted to result in loss or disruption of the phosphatase domain, are associated with a severe phenotype and loss of independent ambulation by the early twenties. Whereas the few reported missense mutations and also those truncating mutations occurring at the C-terminus after the phosphatase domain cause a rather mild phenotype and patients were still ambulatory above the age 30 years. Charcot-Marie-Tooth neuropathy and Centronuclear Myopathy causing mutations have been shown to occur in proteins involved in membrane remodeling and trafficking pathway mediated by phosphoinositides. Earlier studies have showing the rescue of MTM1 myopathy by MTMR2 overexpression, emphasize the importance of maintaining the phosphoinositides equilibrium and highlight a potential compensatory mechanism amongst members of this pathway. This proved that the regulation of expression of these proteins involved in the membrane remodeling pathway may compensate each other's loss- or gain-of-function mutations by restoring the phosphoinositides equilibrium. This provides a potential therapeutic strategy for neuromuscular diseases resulting from mutations in the membrane remodeling pathway.

The heterozygous melanocortin 4 receptor (MC4R) mutation is considered a primary factor in 2% to 4% of patients with obesity. Pharmacological treatment, including long-term, is emerging as a new standard for obese patients; however, there are no published reports on treatment outcomes in obese patients with documented MC4R mutations. Weight loss occurs with diet restriction or bariatric surgery and is often followed by a weight regain phase in patients with or without the MC4R mutation, indicating the need for an alternative approach to weight loss such as pharmacological treatment.This case study describes a 48-year-old woman who experienced lifelong obesity and a history of dieting that started in childhood with episodes of weight loss followed by weight gain. Her highest weight was 320 pounds in 2007. Her family's medical history included diabetes, prediabetes, and obesity. She presented to the clinic in January 2013 weighing 286.2 pounds with at-risk cardiovascular and cardiometabolic profiles. Treatment was initiated to control insulin resistance and metabolic syndrome, lower cholesterol and triglycerides, and manage hypothyroidism. Weight-loss treatment also began with a combination of bupropion and naltrexone or carbonic anhydrase inhibitors and naltrexone. Her on-treatment nutrition included eating from all food groups without counting calories or excessive exercise. In late 2014, she tested positive for a heterozygous complete loss-of-function MC4R gene mutation and weighed 213 pounds. The patient's lowest weight while on treatment was 166 pounds in August 2015. By November 2016, after experiencing partial weight regain, her weight had been stable for 6 months at approximately 198 pounds,a 30% reduction in weight from 2013.Pharmacological treatment is emerging as a recommended approach to weight loss. This case is an example of how, despite partial weight regain after weight loss, targeted pharmacological treatment can improve long-term outcomes including in patients with the MC4R complete loss-of-function mutation.

Background: Post Roux-en-Y gastric bypass (RYGB) hyperinsulinemia hypoglycemia (HH) is presumably related to enhanced glucagon-like peptide-1 (GLP-1) response with partial pancreatectomy as recommended treatment. A new approach successfully uses short-acting GLP-1 mimetics, challenging assumptions about post-RYGB HH etiology and standard treatment.Case Description: A 61-year-old woman with lifelong obesity leading to RYGB at 304 pounds followed by 10 years of fatigue and weight fluctuation initially lost 100 pounds, regained 80 pounds, then lost 60 pounds with aggressive diet and exercise. Ten years post-RYGB, she presented with extreme fatigue and weight regain. Mixed-meal tolerance testing revealed post prandial HH: 30-minute post-prandial insulin response of 550.8 mU/mL; 60-and 90-minute post-prandial glucose of 48 and 56 mg/dL, respectively; weight of 219 pounds, BMI 36.4. Initial treatment included pre-meal acarbose 50 mg and exenatide 5 µg three times/day, balanced meals and snacks, and no prescribed exercise. Insulin and glucose levels improved immediately accompanied by weight loss. Acarbose and exenatide doses were adjusted until the insulin response stabilized. Zonisamide (50 mg) and naltrexone (25 mg) twice/day were also prescribed after detecting elevated agouti-related peptide. By 12 months, the patient's weight was 157 pounds (28% weight loss) and BMI 26.1. By 18 months, her weight was 140 pounds (36% weight loss) and BMI 23.3. Conclusions:This report is the first to describe effective use of short-acting exenatide combined with an alpha-glucoside inhibitor to regulate post-RYGB post-prandial HH and facilitate concomitant weight loss, and it challenges previous assumptions regarding post-prandial HH etiology and invasive treatment with partial pancreatectomy.