The Mystery of LVB

1. The Mystery of LVB Susan A. Bender Summer Research Institute 2014 sbender@jackson.k12.ms.us 601-955-9425

2. The Mystery of LVB: The Story Begins • A 56 year old man presents to his physician in early December with chills, fever, upset stomach, and right sided chest pain. • Within the last two weeks, he noted a loss of appetite, diarrhea, weight loss, increased thirst, jaundice, and severe swelling of the lower extremities. • Prior to visiting his physician, he had been visiting his brother’s country home in Germany and enjoying working in the winter air from dawn until dusk. • As his symptoms worsened, he decided to cut his trio short and returned to Vienna in his open air carriage.

3. The Mystery of LVB: Preliminary Diagnosis • Based on the initial description of the patient, what do you believe could be the source of his symptoms? • On what evidence do you base your conclusion? • What additional information would you need to proceed? • What questions about family medical history and prior medical conditions in the patient might be useful? • Would your conclusions and treatment options be different if instead of 2013, this patient presented to you in the 1770’s?

4. The Mystery of LVB: The Physical Exam • Communication was difficult because the patient was deaf. • Exam revealed a stocky powerfully built mad who was acutely ill. • His skin was hot and flushed and he was having great difficulty breathing. • There was marked swelling in both lower extremities. • There were signs of jaundice.

5. The Mystery of LVB: Diagnosis Continued • Does this additional information change your conclusions? • How would you treat this patient?

6. The Mystery of LVB: The Diagnosis • The patient was diagnosed with pneumonia and edema. • Palpation of he abdomen revealed rebound tenderness and an acutely enlarged liver.

7. The Mystery of LVB: The Treatment • The patient was treated with a series of herbal diuretics which proved to be ineffective. • Drains were inserted to remove abdominal fluid and initially removed 11 liters. • In an attempt to remove addition fluid, the patient was provided with iced alcoholic punch. While this allowed the patient to sleep, it took several days to begin to flush the toxins from his system. • His battle with pneumonia and fluid retention lasted for three months. • In the final stages of his life, he became anemic, comatose, and died.

8. The Mystery of LVB: Treatment Today • Given the fact that there were no antibiotics in the 1770’s. • How would you treat the patient today? • How might the outcome have been different wit the advent of modern treatment practices?

9. The Mystery of LVB: Patient Medical History • The patient had suffered his entire life with several fairly debilitating illnesses. • The worst of which was progressive hearing loss that began in his 20’s and was complete by his early 40’s. • His deafness was accompanied by bouts of depression and alcoholism that lasted for the remainder of his life. • He also was plagued by prolonged periods of abdominal pain, diarrhea and constipation. • As a child, the patient had smallpox, typhus, and asthma. • In his late 40’s he noted chronic headaches and joint pain, jaundice and bleeding from the rectum. • The patient was never married, but according to friends was never “without love”

10. The Mystery of LVB: Diagnosis Continued • Does this additional information change your conclusions? • How would you treat this patient?

11. The Mystery of LVB: Family Medical History • Patients alcoholic father died at age 52 of unknown causes. • His mother died at age 35 of tuberculosis. • Three of his siblings died before the age of two. • Another sibling, a brother, died at the age of 41 of poisoning. • His eldest brother died at 72 of heart related complications.

12. The Mystery of LVB: Diagnosis Continued • Does this additional information change your conclusions?

13. The Mystery of LVB: The Autopsy • The autopsy revealed dilation of the auditory nerves. Examination also revealed the presence of arteriosclerosis, (plaque), in the auditory arteries • There is also a continuing dispute about the possible other causes of death: • alcoholic cirrhosis, • typhus, • infectious hepatitis, • lead poisoning • syphilis • sarcoidosis • Whipple's disease • Use your electronic devices to research each of the diseases what are the symptoms of each? How are these symptoms indicated to determine the validity of your diagnosis before you proceed with modern tests on the remains of the patient

14. The Mystery of LVB: Modern Scientific Tests • The evidence suggests that the deafness may be directly related to tertiary syphilis. • The remainder of the symptoms may be attributable to complications of lead poisoning • There are skull fragments that still exist from the remains that are positively identified as belonging to the patient. • There are also hair samples that may belong to the patient that can be tested with modern techniques. • You will perform a mitochondrial DNA test on the available forensic evidence to determine the identity of the donor of the hair and will test the skull fragments for the presence of lethal levels of lead

15. The Mystery of LVB: The Conclusions • MATERIALS • DNA sequence from skull fragment of Beethoven • DNA sequence from Beethoven’s Mother • DNA sequence from hair believed to belong to Beethoven • DNA sequence from Beethoven’s Father • DNA sequence standard to ensure the tests accuracy

16. The Mystery of LVB: The Conclusions 2 • Procedure: • Review your data table and you will see the labels that should be attached to create 5 separate columns, as seen in the example below.

17. With your PENCIL you are going to simulate the action of a restriction enzyme. Scan your DNA strips until you find the letters “GG CC”. MARK across the strip between the letter G and C, you will be forming a fragment that ends with a GG and another that begins with a CC. The Mystery of LVB: Conclusions 3 • MAKE SURE THAT YOU KEEP EACH PERSON’S DNA SEPARATE THROUGHOUT THIS ACTIVITY. • To simulate the action of radioactive probes use a highlighter and cover the letters CAT in each of your segments • With your PENCIL you are going to simulate the action of a restriction enzyme. Scan your DNA strips until you find the letters “GG CC”. MARK across the strip between the letter G and C, you will be forming a fragment that ends with a GG and another that begins with a CC.

18. The Mystery of LVB: Conclusions 4 • Count the number of bases in each fragment, (the number of letters). Match this number to the numbers in the chart provided. • Then draw a line in the box that corresponds to the number of letters in that piece. Use your highlighter to mark the position of each of the CAT sequences in each piece on the line that you have drawn.

19. The Mystery of LVB: Diagnosis Continued • Now you will repeat this process for each of the other fragments of DNA that you have created. • You will compare the DNA sequences from the skull fragments known to belong to Beethoven to the hair sample whose donor is still in question. You are attempting to determine if the hair and the skull fragments are from the same individual in this case Ludwig van Beethoven. • How will you know if the DNA samples are a match? How can you be sure that the samples are from the same individual? What further tests might you be able to conduct to determine the cause and manner if death for Beethoven?

20. The Mystery of LVB: Test for LEAD You will now perform a confirmatory test to determine the presence of lead in several of the skull fragment samples collected after the death of Beethoven. This is a colorimetric test that will measure the ppm (parts per million) or lead in each of the skull fragment samples, • You will perform a colorimetric test for the presence of lead in the skull fragments. Bear in mind that once you have used a portion of the sample for the test you have destroyed a portion of the skull fragment. So use the smallest samples possible to conduct your test. • Break off three small pieces from each of the skull fragments that have been provided to you. • In separate small micro-centrifuge tubes grind each of the skull fragments into a powder. • Add a drop of the lead indicator solution to each of the tubes and mix thoroughly. • If the sample changes color …this is a positive test for the presence of lead. The darker the color the greater the concentration. See the chart below.

21. The Mystery of LVB: Lead Comparison Chart • Based on the results of your testing, did the patient suffer form lead poisoning? • From your research, what is the significance of this data? Could this account for all of the patients symptoms?