Pediatric Genetics & Genomics

Lab Testing and Genetic & Metabolic Disease: Spotting the "Red Flags"

April 28, 2020

1. Pediatric Genetics and Genomics April 28, 2020 Lab Testing and Genetic & Metabolic Disease: Spotting the "Red Flags" 1 Leah Burke, MD University of Vermont Mark Korson, MD VMP Genetics, LLC

2. Get the Most Out of Your Experience Use the Q&A Button to submit questions during today’s session Recording and slides will be sent by email For further information, contact [email protected] 2

3. Founding year: 1972 Hubs/Locations: 15/210 Patients per year: 100,000 The Weitzman Institute is a program of 3

4. 4 The Weitzman Institute works to improve primary care and its delivery to medically underserved and special populations through research , innovation , and the education and training of health professionals. 4 Genetics | 2/18/20 4

5. New England Regional Genetics Network The NERGN project is supported by the Health Resources and Services Administration (HRSA) of the U.S. Department of Health and Human Services (HHS) under grant number UH7MC30778; New England Regional Genetics Network; total award amount: 1.5 million; 100% from governmental sources. This information or content and conclusions are those of the author and should not be construed as the official position or policy of, nor should any endorsements be inferred by HRSA, HHS or the U.S. Government. 5

6. CME Credits Available CME credits available to live webinar participants only A brief survey will be sent after this session to those who registered and attended this webinar CME certificate will be sent to participants who complete the survey American Academy Family Physicians (AAFP) CME Credit 6

7. Disclosures No Disclosures 7

8. Today’s Presenters Dr. Leah W. Burke, MD Dr. Mark Korson, MD 8

9. Goals Review the genetic and genomic testing modalities Identify the clinical and family history findings that raise a concern for a genetic disorder Review how simple biochemical tests that can determine the competence of human metabolism Describe a few findings on routine biochemical testing that can increase suspicion about an underlying metabolic disease

10. Genetic Testing: Screening vs. Diagnostic Testing done on a particular population Individuals are asymptomatic Not designed to diagnose, simply to identify individuals at a higher risk May lead to diagnostic tests Testing done on individuals Individuals often symptomatic Individuals may have had a positive screening test May lead to treatment options

11. Diagnostic Genetic Testing Karyotype FISH Image F luorescence I n S itu H ybridization Microarray Jax.com

12. Karyotype

13. Karyotype Compare the Karyotype to published Idiograms 2q34 2q10 2p24

14. Diagnostic Genetic Testing Karyotype FISH Image F luorescence I n S itu H ybridization Microarray Jax.com

15. FISH : Fluorescence in situ Hybridization

16. Diagnostic Genetic Testing Karyotype FISH Image F luorescence I n S itu H ybridization Microarray Jax.com

17. What’s in a name? A test by any other name… Microarray Chromosomal microarray Whole genome microarray Comparative Genomic Hybridization (CGH) Array CGH Function is to determine copy number variants

18. Microarray Duplication in patient Deletion in patient Normal copy number

19. 20 Karyotype Individual FISH test Microarray Can detect whole chromosome differences, translocations, and large deletions or duplications Only looks at specific areas for deletions or duplications Can detect very small duplications or deletions

20. What about sequencing? 21

21. Sanger Sequencing

22. A massively parallel sequencing technology in which millions of overlapping “reads” of DNA sequences are done simultaneously Creates an enormous amount of data to be analyzed Can detect single gene mutations including nonsense, missense, splice-site, and frame-shift mutations 23 Next Generation Sequencing

23. 24

24. 25

25. Whole Exome Sequencing (WES) Now being offered clinically Parallel sequencing of at least 98% of the coding sequences Whole Genome Sequencing (WGS) Parallel sequencing of the coding and non-coding sequences Some conditions are caused by DNA changes outside the “gene” 26 Both are being considered in rapid testing of sick newborns in the NICU and other places

26. Lots of data…What happens next? 27

27. 28 Variants of Uncertain Significance: The Dreaded VUS

28. With whole genome sequencing or whole exome sequencing, the chance of VUS is even greater than with microarray testing The reading of the variants can be different between different labs and over time 29 Variants of Uncertain Significance: The Dreaded VUS

29. So when should you refer for a genetics/metabolism evaluation? 30

30. REMEMBER THE RULE OF “TWO/TOO” (when asking questions) TOO tall TOO short TOO early TOO many TOO young TOO different TWO tumors TWO generations TWO in the family TWO birth defects

31. Let’s start with the Too’s TOO tall TOO short TOO early TOO many TOO young TOO different 32

32. Acromelic Mesomelic Rhizomelic

33. Too tall Cerebral gigantism ( Sotos syndrome) Marfan syndrome Beckwith- Weidemann syndrome Homocystinuria XYY males Other syndromes

34. Prenatal onset overgrowth Cerebral gigantism Dolichocephaly Frontal bossing, prognathism , pointed chin, downslating palpebral fissures High-arched palate Developmental delay, poor coordination, behavioral problems, autism, seizures Ventriculomegaly , absent corpus callosum, persistent cavum septum pellucidum Sotos syndrome

35. Marfan Syndrome Tall stature with long bone overgrowth Long narrow face High-arched palate Subluxation of lenses Spontaneous pneumothoraces Arachnodactyly Aortic root dilation and dissection Mitral regurgitation Scoliosis Pectus deformities Joint hypermobility Striae dystrophica Recurrent hernias Usually due to a FBN1 (fibrillin 1) mutation Also consider TGFBR1 and TGFBR2

36. Beckwith-Wiedemann LGA babies without maternal diabetes Macroglossia Hemi-hypertrophy Ear creases and or posterior pits Umbilical hernia or omphalocele Wilms tumor

37. Too short History of Growth deficiency Onset Prenatal – intrauterine growth retardation Postnatal Growth maturation Normal Delayed Proportional Proportions are normal Malproportion

38. Achondroplasia Short-limb dwarfism – rhizomelic shortening Macrocephaly with frontal bossing Midface hypoplasia Foramen magnum stenosis Upper airway obstruction Kyphosis and lumbar lordosis Trident hand Limited elbow extension Generalized joint hypermobility Bowing of legs Conductive hearing loss Delayed motor development Occasional hydrocephalus Mutations in FGFR3 gene (80% de novo)

39. Too many 46

40. Too many 47

41. Skin Manifestations: Subcutaneous Neurofibromas

42. Too different 49

43. Eyes Too close Normal Too wide Short Normal Long Upslanting Nl Downslanting Ears

44. Too different Developmental delay Autism 51

45. Initial Genetics Evaluation: First Tier Three generation family history Medical history for clues about birth/environmental causes Physical evaluation for signs of underlying syndromes Targeted testing for a specific syndrome Metabolic and/or mitochondrial testing if indicated Chromosomal microarray Fragile X DNA testing

46. Fragile X physical features Macrocephaly Long face with prominent jaw and long ears Joint hypermobility May not have any striking features

47. Fragile X Syndrome Caused by a change in the FMR1 gene located on the X chromosome A full mutation is expressed in 100% of males and 50% of females Intellectual disability is a major feature in affected males Must be tested for with a specific test that determines the repeat size

48. Initial Genetics Evaluation: First Tier Three generation family history Medical history for clues about birth/environmental causes Physical evaluation for signs of underlying syndromes Targeted testing for a specific syndrome Metabolic and/or mitochondrial testing if indicated Chromosomal microarray Fragile X DNA testing

49. Chromosomal Microarray An important part of the First Tier Numerous pathogenic copy number variants (CNVs) have been identified in the etiology of autism Yield is thought to be about 10% Several “hot spots” or recurrent CNVs have been identified Often present in unaffected parents – variable penetrance

50. Case 18 month old girl referred to genetics for gross motor delays Additional features Small VSD Epicanthal folds and puffy eyelids Upturned nose and wide mouth Difficulty gaining weight Facial features reminiscent of Williams syndrome but didn’t completely fit Did the first tier of testing 57

51. Case Seen again at 5 ½ years and proposed doing exome sequencing Received approval from insurance, but parents decided to wait At 6 years of age, she was diagnosed with celiac disease Parents proceeded with testing A pathogenic variant was found in SETD5 58

52. SETD5 Associated with an autosomal dominant neurodevelopmental syndrome Found on the short arm of chromosome 3 Deletions in this area have been known to cause a neurodevelopmental syndrome 59

53. 60 Rarechromo.org

54. REMEMBER THE RULE OF “TWO/TOO” (when asking questions) TOO tall TOO short TOO early TOO many TOO young TOO different TWO tumors TWO generations TWO in the family TWO birth defects

55. The Two’s 62

56. Chief complaint Family History, if available: Include 3 generations if available Designate how each person is related Include developmental history Do not limit to symptoms found in patient Include causes of death, esp. in early deaths Include miscarriages & early infant deaths Include consanguinity and ethnicity WHAT THE PCP SHOULD PROVIDE WITH THE REFERRAL

57. (35) Breast Cancer @ 63 died @ 67 - heart disease Breast Cancer @ 58 post-menopausal died at 63 - metastases Breast cancer @ 80 died @ 83 Irish, English, Scottish Irish, German (60) (87) (62) Prostate cancer 70’s (84) (62)

58. Russian Jewish (35) bre ca @ 42 died @ 48 bre ca @ 45 died @ 52 bil bre ca @ 45, 52 died @ 80

59. SC – s ome c oncerns re: diseases in family “Do you have any questions about diseases or conditions that run in your family?” R – r eproduction problems “Have there been any problems with pregnancy, infertility, or birth defects in your family?” E – e arly disease, death, or disability “Have any members of your family become sick or died at an early age?” E – e thnicity “How would you describe your ancestry?” OR “What countries do your families originate from? N – n on-genetic factors “Are there any other nonmedical conditions that run in your family?” FAMILY HISTORY SCREEN MNEMONIC

60. Let’s take a walk to the Chemistry Lab…

61. BASIC LAB TESTING: THE THEORY

62. Can help assess the competence of metabolism of: Protein Carbohydrates Fat BASIC LAB TESTING: THE THEORY

63. Can help assess the competence of metabolism of: Protein Carbohydrates Fat BASIC LAB TESTING: THE THEORY

64. - C - N - - - - ANATOMY OF AN AMINO ACID

65. - C - N - - - - UREA CYCLE

66. - C - - - ORGANIC ACID

67. AMINO ACIDS CHANGE Krebs cycle Urea cycle ORGANIC ACIDS NH 3 ATP ELECTRON TRANSPORT CHAIN PROTEIN DEGRADATION

68. AMINO ACIDS CHANGE Krebs cycle Urea cycle ORGANIC ACIDS NH 3 ATP ELECTRON TRANSPORT CHAIN PROTEIN DEGRADATION DEFECTS Organic acidemia Urea cycle disorder

69. AMINO ACIDS CHANGE Krebs cycle Urea cycle ORGANIC ACIDS NH 3 ATP ELECTRON TRANSPORT CHAIN PROTEIN DEGRADATION TESTING Organic acidemia Urea cycle disorder

70. AMINO ACIDS CHANGE Krebs cycle Urea cycle ORGANIC ACIDS NH 3 ATP ELECTRON TRANSPORT CHAIN PROTEIN DEGRADATION TESTING - Ammonia - Blood gases - Electrolytes - Anion gap Organic acidemia Urea cycle disorder

71. BLOOD GAS MEASUREMENT

72. BLOOD GAS MEASUREMENT Blood pH=7.4 Low pH  acidic High pH  alkalotic Blood HCO 3 =24 Low HCO 3  acidic High HCO 3  alkalotic

73. TACHYPNEA  A WAY TO BLOW OFF ACID H + + HCO 3 - H 2 CO 3 H 2 O + CO 2

74. TACHYPNEA  A WAY TO BLOW OFF ACID Blood pH=7.4 Low pH  acidic High pH  alkalotic Blood pCO 2 =40 Low pCO 2  rapid breathing High pCO 2  slower breathing Blood HCO 3 =24 Low HCO 3  acidic High HCO 3  alkalotic

75. BLOOD GAS MEASUREMENT

76. BLOOD GAS MEASUREMENT 1 ° METABOLIC ACIDOSIS

77. BLOOD GAS MEASUREMENT 1 ° METABOLIC ACIDOSIS

78. BLOOD GAS MEASUREMENT 1 ° METABOLIC ACIDOSIS 2° RESPIRATORY ALKALOSIS

79. BLOOD GAS MEASUREMENT 1 ° METABOLIC ACIDOSIS 2° RESPIRATORY ALKALOSIS

80. BLOOD GAS MEASUREMENT

81. BLOOD GAS MEASUREMENT 2° RESPIRATORY ALKALOSIS

82. BLOOD GAS MEASUREMENT 2° RESPIRATORY ALKALOSIS

83. BLOOD GAS MEASUREMENT 2° RESPIRATORY ALKALOSIS 1 ° METABOLIC ACIDOSIS

84. BLOOD GAS MEASUREMENT 1 ° RESPIRATORY ALKALOSIS 2 ° METABOLIC ACIDOSIS THIS SLIDE HAS BEEN UPDATED POST PRODUCTION. THE INFORMATION ON THIS SLIDE IS CORRECT. THE INFORMATION ON THE SLIDE SHOWN ON THE RECORDING IS INCORRECT AS POINTED OUT BY DR. KORSON

85. BLOOD GAS MEASUREMENT

86. A CASE OF VOMITING & LETHARGY A 10 month old girl presents to the ER with her second bout of vomiting and dehydration. The first resolved with IV fluids. She is now lethargic and tachypneic. Her blood gases show- pH=7.54, pCO 2 =16, HCO 3 =17. Electrolytes reveal Na=142, K=3.1, Cl=107, HCO 3 =18. Glucose measures 62 mg/dL.

87. QUESTION Which ONE of the following choices regarding the patient’s presentation is the most diagnostically helpful? The glucose level This is the second episode of vomiting/dehydration The respiratory alkalosis The metabolic acidosis The patient’s lethargy

88. QUESTION Which ONE of the following choices regarding the patient’s presentation is the most diagnostically helpful? The glucose level This is the second episode of vomiting/dehydration The respiratory alkalosis The metabolic acidosis The patient’s lethargy

89. RESPIRATORY ALKALOSIS Hyperventilation Medications/drugs Pain Fever Intracranial: Malformations Trauma Hyperammonemia Causes

90. THE ANION GAP Difference between measured anions and cations in the blood Definition Calculation Anion gap = Na - (Cl + HCO 3 ) Anion gap = 140 - (105 + 25) Anion gap = 10 Normal anion gap = 10-15

91. NON-ANION GAP ACIDOSIS Anion gap = Na - ( Cl + HCO 3 ) Anion gap = 140 - (115 + 10) Anion gap = 15  Bicarbonate loss (urine or stool) HIGH NORMAL LOW

92. ANION GAP ACIDOSIS Anion gap = Na - ( Cl + HCO 3 ) Anion gap = 140 - (105 + 10) Anion gap = 25  Anion accumulation NORMAL HIGH LOW

93. AMINO ACIDS CHANGE Krebs cycle Urea cycle ORGANIC ACIDS NH 3 ATP ELECTRON TRANSPORT CHAIN PROTEIN DEGRADATION DEFECTS: Ammonia Blood gases Electrolytes Anion gap Organic acidemia Urea cycle disorder

94. Can help assess the competence of metabolism of: Protein Carbohydrates Fat BASIC LAB TESTING: THE THEORY

95. CARBOHYDRATE ANABOLISM Krebs cycle ATP ELECTRON TRANSPORT CHAIN LACTATE PYRUVATE GLUCOSE GLYCOGEN GLYCOLYSIS

96. CARBOHYDRATE CATABOLISM LACTATE PYRUVATE GLUCOSE GLYCOGEN GLUCONEOGENESIS

97. CARBOHYDRATE CATABOLISM GLYCOGEN STORAGE DISEASE LACTATE PYRUVATE GLUCOSE GLUCONEOGENESIS GLYCOGEN

98. CARBOHYDRATE CATABOLISM DEFECTS IN GLUCO-NEOGENESIS LACTATE PYRUVATE GLUCOSE GLUCONEOGENESIS GLYCOGEN

99. CARBOHYDRATE CATABOLISM TESTING LACTATE PYRUVATE GLUCOSE GLUCONEOGENESIS GLYCOGEN

100. CARBOHYDRATE CATABOLISM TESTING - Glucose - Lactate LACTATE PYRUVATE GLUCOSE GLUCONEOGENESIS GLYCOGEN

101. Can help assess the competence of metabolism of: Protein Carbohydrates Fat BASIC LAB TESTING: THE THEORY

102. Krebs cycle ATP ELECTRON TRANSPORT CHAIN FATTY ACID CATABOLISM LIVER FATTY ACIDS KETONES ACETYL CoA Four-step oxidative cycle

103. Krebs cycle ATP ELECTRON TRANSPORT CHAIN FATTY ACID CATABOLISM PERIPHERY KETONES ACETYL CoA

104. Krebs cycle ATP ELECTRON TRANSPORT CHAIN FATTY ACID OXIDATION DEFECTS FATTY ACIDS KETONES ACETYL CoA Four-step oxidative cycle

105. Krebs cycle ATP ELECTRON TRANSPORT CHAIN FATTY ACID OXIDATION TESTING FATTY ACIDS KETONES ACETYL CoA Four-step oxidative cycle

106. Krebs cycle ATP ELECTRON TRANSPORT CHAIN FATTY ACID OXIDATION TESTING - Ketones FATTY ACIDS KETONES ACETYL CoA Four-step oxidative cycle

107. Aketosis or hypoketosis is always an abnormal response to hypoglycemia (or even very prolonged fasting) Exception – newborns HYPOGLYCEMIA

108. Routine laboratory testing can be a good indicator for assessing basic intermediary metabolism: Abnormalities might suggest an underlying acute metabolic disorder SUMMARY Blood gases Electrolytes, bicarb (anion gap) Ammonia Glucose Lactate Urinalysis

109. WHICH IS WHICH? Which set of lab results most likely correlates with diarrhea and which with a metabolic disorder? A CASE 1 – diarrhea CASE 2 – metabolic disorder B CASE 1 – metabolic disorder CASE 2 – diarrhea ANALYTE CASE 1 CASE 2 NORMALS Sodium 141 135 133–146 Potassium 5 5 3.7-5.9 Chloride 116 103 98-107 Bicarbonate 10 10 21-31 Anion gap 15 22 8-15 (without K)

110. WHICH IS WHICH? Which set of lab results most likely correlates with diarrhea and which with a metabolic disorder? A CASE 1 – diarrhea CASE 2 – metabolic disorder B CASE 1 – metabolic disorder CASE 2 – diarrhea ANALYTE CASE 1 CASE 2 NORMALS Sodium 141 135 133–146 Potassium 5 5 3.7-5.9 Chloride 116 103 98-107 Bicarbonate 10 10 21-31 Anion gap 15 22 8-15 (without K)

111. NON-ANION GAP ACIDOSIS Anion gap = Na - ( Cl + HCO 3 ) Anion gap = 140 - (115 + 10) Anion gap = 15  Bicarbonate loss (urine or stool) HIGH NORMAL LOW

112. ANION GAP ACIDOSIS Anion gap = Na - ( Cl + HCO 3 ) Anion gap = 140 - (105 + 10) Anion gap = 25  Anion accumulation NORMAL HIGH LOW

113. THE INFANT WITH LIVER DISEASE A 6-month old infant male, product of a healthy pregnancy/labor/delivery, was found to have a distended belly at 3 months. By that time, he had lost 1 kg of weight and was failing to thrive. He continues to feed every 3-4 hours day and night. The liver was noted to be enlarged at 5 months of age. He was evaluated in the ED shortly after that for fever and upper respiratory symptoms.

114. THE INFANT WITH LIVER DISEASE Lab tests: Glucose=39 mg/dL Mild metabolic acidosis: HCO 3 =15 ALT=118, AST=274 Other liver functions normal Lactate=3.6 mmol/L (NL<2.2) Urinalysis: pH=6.5 Based on these clinical/lab data, this patient has liver disease. Which other organ are you worried about? Brain Kidneys Distal extremities Heart Muscle

115. THE INFANT WITH LIVER DISEASE Lab tests: Glucose=39 mg/dL Mild metabolic acidosis: HCO 3 =15 ALT=118, AST=274 Other liver functions normal Lactate=3.6 mmol/L (NL<2.2) Urinalysis: pH=6.5 Based on these clinical/lab data, this patient has liver disease. Which other organ are you worried about? Brain Kidneys Distal extremities Heart Muscle

116. May be associated with: Pan-liver dysfunction Cholestasis Just hepatomegaly Often associated with renal tubular d ysfunction (renal Fanconi syndrome) METABOLIC LIVER DISEASE

117. A CASE OF HYPOGLYCEMIA A 2 ½ year old boy develops a cough and cold symptoms. His PCP diagnoses a pharyngitis; the patient takes only smaller volumes than usual. On the third day, he is pale and difficult to rouse. He is rushed to the ED and has a seizure in the car. His blood glucose measures 25 mg/dL. Blood gases – pH=7.29, pCO 2 =31, HCO 3 =15. Electrolytes measure Na=131, K=4.4, Cl=99. His urinalysis shows - pH=5.0, no glucose or protein, and 1+ ketones.

118. QUESTION Which ONE of the following choices regarding the patient’s presentation is the most diagnostically helpful? Hypoglycemia Metabolic acidosis Hypoglycemia with seizure Hypoglycemia with 1+ urine ketones Hypoglycemia, hyponatremia, with seizures

119. QUESTION Which ONE of the following choices regarding the patient’s presentation is the most diagnostically helpful? Hypoglycemia Metabolic acidosis Hypoglycemia with seizure Hypoglycemia with 1+ urine ketones Hypoglycemia, hyponatremia, with seizures

120. HYPOKETOTIC HYPOGLYCEMIA

121. HYPOKETOTIC HYPOGLYCEMIA FAT FATTY ACIDS KETONES HIGH Insulin State

122. HYPOKETOTIC HYPOGLYCEMIA HIGH Insulin State Insulin tumor Infant of DM mother Beckwith-Wiedemann syndrome Iatrogenic

123. HYPOKETOTIC HYPOGLYCEMIA FAT FATTY ACIDS KETONES LOW Insulin State

124. HYPOKETOTIC HYPOGLYCEMIA LOW Insulin State Fatty acid oxidation defect Glycogen storage disease type I

125. Aketosis or hypoketosis is always an abnormal response to hypoglycemia (or even very prolonged fasting) Exception – newborns HYPOGLYCEMIA

126. THE CRITICALLY ILL INFANT A male is the 5th child of non-consanguineous parents following a normal pregnancy and delivery. Within 12 hours, he developed respiratory distress and lethargy, requiring intubation and ventilation. Blood gases showed: pH=7.21, pCO 2 =10, HCO 3 =4. Electrolytes: Na=137, K=4.9, Cl=105, measured HCO 3 =4. Glucose=44 mg/dL. The patient was dehydrated; lactate measured 21 mmol/L (NL<2.2) but persisted despite rehydration. Ammonia=105 μ mol/L. Urinalysis: ketones=3+.

127. QUESTION The most likely reason for the high lactic acid level in this patient is dehydration leading to poor perfusion? True False

128. QUESTION The most likely reason for the high lactic acid level in this patient is dehydration leading to poor perfusion? True False

129. THE CRITICALLY ILL INFANT A male is the 5th child of non-consanguineous parents following a normal pregnancy and delivery. Within 12 hours, he developed respiratory distress and lethargy, requiring intubation and ventilation. Blood gases showed: pH=7.21, pCO 2 =10, HCO 3 =4. Electrolytes: Na=137, K=4.9, Cl=105, measured HCO 3 =4. Glucose=44 mg/dL. The patient was dehydrated; lactate measured 21 mmol/L (NL<2.2) but persisted despite rehydration. Ammonia=105 μ mol/L. Urinalysis: ketones=3+.

130. Questions? Call Genetics! We like answering your questions! THANK YOU FOR PARTICIPATING TODAY.