CLINICAL REPORT
Biochemical Abnormalities in Pearson Syndrome Beatrice Letizia Crippa,1,2 Eyby Leon,1 Amy Calhoun,1,6 Amy Lowichik,3,4 Marzia Pasquali,1,3,5 and Nicola Longo1,3,5* 1
Department of Pediatrics, Division of Medical Genetics, University of Utah, Salt Lake City, Utah
2
University of Milano, Milan, Italy Department of Pathology, University of Utah, Salt Lake City, Utah
3 4
Primary Children’s Hospital, Salt Lake City, Utah
5
ARUP Laboratories, University of Utah, Salt Lake City, Utah Department of Pediatrics, Division of Genetics and Metabolism, University of Minnesota, Minneapolis, Minnesota
6
Manuscript Received: 30 September 2014; Manuscript Accepted: 15 December 2014
Pearson marrow-pancreas syndrome is a multisystem mitochondrial disorder characterized by bone marrow failure and pancreatic insufficiency. Children who survive the severe bone marrow dysfunction in childhood develop Kearns–Sayre syndrome later in life. Here we report on four new cases with this condition and define their biochemical abnormalities. Three out of four patients presented with failure to thrive, with most of them having normal development and head size. All patients had evidence of bone marrow involvement that spontaneously improved in three out of four patients. Unique findings in our patients were acute pancreatitis (one out of four), renal Fanconi syndrome (present in all patients, but symptomatic only in one), and an unusual organic aciduria with 3-hydroxyisobutyric aciduria in one patient. Biochemical analysis indicated low levels of plasma citrulline and arginine, despite low-normal ammonia levels. Regression analysis indicated a significant correlation between each intermediate of the urea cycle and the next, except between ornithine and citrulline. This suggested that the reaction catalyzed by ornithine transcarbamylase (that converts ornithine to citrulline) might not be very efficient in patients with Pearson syndrome. In view of low-normal ammonia levels, we hypothesize that ammonia and carbamylphosphate could be diverted from the urea cycle to the synthesis of nucleotides in patients with Pearson syndrome and possibly other mitochondrial disorders. Ó 2015 Wiley Periodicals, Inc.
Key words: Pearson syndrome; mitochondrial disease; organic aciduria; orotic acid; plasma amino acids; uridine
INTRODUCTION Pearson marrow-pancreas syndrome was first described in 1979 as a fatal multisystem mitochondrial disorder that occurs in early infancy [Pearson et al., 1979; DiMauro and Hirano, 1993]. Bone marrow failure is the first typical feature of Pearson syndrome with isolated macrocytic sideroblastic anemia, frequently transfusion-
Ó 2015 Wiley Periodicals, Inc.
How to Cite this Article: Crippa BL, Leon E, Calhoun A, Lowichik A, Pasquali M, Longo N. 2015. Biochemical abnormalities in Pearson syndrome. Am J Med Genet Part A 167A:621–628.
dependent, or in association with thrombocytopenia and neutropenia [Pearson et al., 1979]. The presence of vacuolization in granulous and erythroblastic progenitors, visible on the myelogram, and the presence of ringed sideroblasts are typical feature of Pearson syndrome [Muraki et al., 1997; Pearson et al., 1979]. Dysfunction of the exocrine pancreas due to fibrosis is also present in Pearson syndrome with chronic diarrhea, malabsorption, and failure to thrive [Pearson et al., 1979]. Children who survive the severe bone marrow dysfunction develop Kearns–Sayre syndrome later in life (usually before 20 years of age) [McShane et al., 1991; Muraki et al., 1997]. There is no effective therapy for this condition. Other affected organ systems include the kidney (tubulopathy and aminoaciduria) [Superti-Furga et al., 1993; Atale et al., 2009], liver (hepatomegaly, cytolysis, and cholestasis) [Superti-Furga et al., 1993; Atale et al., 2009], endocrine glands (diabetes mellitus, adrenal insufficiency) [Williams et al., 2012], neuromuscular system, and heart [Krauch et al., 2002]. Identification of mitochondrial DNA rearrangements with a spectrum of mtDNA deletions confirms the diagnosis [Rotig et al., 1995] Pearson syndrome can be difficult to distinguish from other mitochondrial disorders when multiple organs are involved early, Conflict of interest: none. Correspondence to: Nicola Longo, M.D., Ph.D., Division of Medical Genetics, Department of Pediatrics, University of Utah, 2C412 SOM, 50 North Mario Capecchi Drive, Salt Lake City, UT 84132. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): DOI 10.1002/ajmg.a.36939
621
622 but in many cases the pancreatic involvement can reveal the diagnosis [Fellman and Kotarsky, 2011; Honzik et al., 2012]. Patients with Pearson syndrome can have lactic acidemia and abnormal urine organic acids, with methylglutaconic aciduria and increased intermediates of the citric acid cycle [Gibson et al., 1992; Ribes et al., 1993; Superti-Furga et al., 1993]. Alterations in plasma amino acids have also been reported [Ribes et al., 1993], but no extensive studies have been performed in these patients. Here we report on four new patients with Pearson syndrome, some with unique clinical manifestations. Some of these patients had unusual biochemical profiles that might aid in their diagnosis, have utility for clinical monitoring, and lead to novel therapies.
MATERIALS AND METHODS A retrospective chart review of the clinical course and laboratory test results was performed for patients followed in our clinic for Pearson syndrome. Informed consent was provided by patients for DNA testing. Testing for common mtDNA point mutations and the common Pearson syndrome deletion were performed in peripheral blood using standard PCR amplification and restriction enzyme digestion at Baylor College of Medical Genetics Laboratory (Houston, TX). Urine organic acids were analyzed by gas chromatography/mass spectrometry; plasma amino acids by ion exchange chromatography; and plasma acylcarnitine profile by tandem mass spectrometry using standard procedures at ARUP Laboratories, Salt Lake City, Utah. Routine testing was performed in a pediatric hospital using standard procedures and age appropriate reference ranges.
AMERICAN JOURNAL OF MEDICAL GENETICS PART A she became transfusion dependent for both red blood cells and platelets. She developed progressive liver failure with cholestasis with severe intestinal dysfunction at 20 months of age requiring total parenteral nutrition. She died at 23 months of age due to septic shock with multiorgan failure. Laboratory studies collected from her initial presentation at 9 month of age to her death at 23 months indicated chronic l actic acidosis (lactic acid 10 3.8 mmol/L, n ¼ 55, normal 0.7–2.1 mmol/L) and abnormal plasma amino acids with elevated alanine (676 386 mM, range 156–1503, n ¼ 18, normal 240–600 mM), low arginine (38 32 mM, range 11–121, n ¼ 18, normal 40–160 mM), low citrulline (despite some values were collected while the patient was on supplements, 28 63 mM, range 0–275, n ¼ 18, normal 10–60 mM), and low ornithine (33 29 mM, range 9–98, n ¼ 18, normal 20–135 mM). Amino acid values fluctuated depending on citrulline supplementation (200 mg/ kg per day starting at 17 months of life). Ammonia levels were nearly always low, with a single value in the normal range (15.6 13.5 mM, n ¼ 5, normal 21–50 mM). Urine organic acids showed a persistent excretion of lactic, pyruvic, 3-hydroxyisobutyric, and 2-ethyl-3-hydroxy propionic acids. These findings suggested mitochondrial dysfunction. Mitochondrial DNA testing revealed heteroplasmy for a large deletion of mtDNA and diagnosis of Pearson Syndrome was made at 21 months. Bone marrow biopsy at 21 months of age indicated hypoplastic marrow with vacuolated erythroid cells that in the presence of megaloblastic changes were supportive of the diagnosis of Pearson syndrome, despite lack of ring sideroblasts.
Patient 2 CLINICAL REPORT Patient 1 A previously healthy female was diagnosed with failure to thrive at 9 months of age, with weight reaching the 1st centile. At 13 months, she was admitted to the hospital for dehydration due to viral gastroenteritis that required admission to the intensive care unit. She had mild metabolic acidosis (anion gap 9 mmol/L, normal 3–16 mmol/L; CO2 13 mmol/L, normal 18–24 mmol/L) and elevated lactate level (4.4 mmol/L; normal 0.7–2.1 mmol/L). Blood counts were normal except for mild thrombocytopenia (platelets 139, normal 150–400 k/ml) and macrocytosis (mean corpuscular volume 89.2 fl, normal 70–86 fl). She had persistent vomiting and abdominal pain with a cyst in the pancreatic tail, normal appearance of the pancreas, and elevated lipase (1,639 U/L, normal 100–150 U/L) prompting a diagnosis of acute pancreatitis. During admission, she developed pancytopenia that improved spontaneously and was initially attributed to viral suppression. She had recurrent pancreatitis 3.5 months later with a peak lipase of 2,662 U/L and then four more times (peak lipase 2,908 U/L) with no major alterations of her blood counts. She was found to have exocrine pancreatic failure at 17 months. Renal tubulopathy was evidenced by persistent aminoaciduria, glycosuria, and tubular acidosis. At 18 months of age, she developed hypotonia, but her brain MRI was normal with normal spectroscopy. Her speech development was normal. Anemia progressively worsened and
A 6-month-old male presented with partial seizures and failure to thrive. He had multiple hospitalizations starting at 8 months of age for renal tubular acidosis, symptomatic hypocalcemia with tetanus, failure to thrive, recurrent infections, dehydration, and pancytopenia requiring blood transfusions. Laboratory studies indicated mildly elevated liver enzymes, alkaline phosphatase, and aldolase (12.2 U/L, normal 3.4–11.6 U/L), with normal urine amino acids (at an outside laboratory) and ammonia levels. He had multiple biopsies, including muscle that showed increased connective tissue suggestive of myopathy; his liver showed mild acute hepatitis with focal fibrosis and patchy steatosis; and his bone marrow was hypoplastic with macrocytosis, anisocytosis, and decreased iron storage. He had mild motor and speech delays. At 2.5 years of age, his height and weight were below the 3rd centile. He was talking in sentences and his muscle tone was mildly reduced. He was receiving sodium citrate, carnitine, calcium carbonate, and iron. At 3 years of age, he began to have daily emesis and diarrhea. He also developed multiple dental abscesses requiring surgical drainage. He was hospitalized twice and found to have severe lactic acidosis with normal anion gap. At 3 years 8 months, urine amino acids showed severe generalized aminoaciduria, proteinuria (protein/creatinine ratio 2.2, normal 0–0.2 mg/mg creatinine; albumin/creatinine ratio 707–720, normal 10–150 mg/g creatinine), suggesting impaired renal tubular function. Plasma amino acids showed mildly elevation of alanine (658 mM/L, normal 240–600 mM), low arginine (30 mM, normal 40–160 mM), normal citrulline (15 mM); urine
26.2 30 20.7 15.2 26.1
6 Pancytopenia Pancreatic insufficiency (8) Not involved Liver dysfunction (6) Tubulopathy (6) Not affected Yes Yes Simultaneous involvement of bone marrow, exocrine pancreas, liver and kidney Failure to thrive Normal Vacuolated erythroid/myeloid precursors, ring sideroblasts Not done Alive (2 years 8 months) 12 Anemia, thrombocytopenia, neutropenia Pancreatic insufficiency (36) DM type 1 (30) Liver dysfunction (24), cholelithiasis (30) Tubulopathy (30) Not affected No Yes DM type 1, late-onset pancreatic exocrine dysfunction Failure to thrive Normal Vacuolated erythroid/myeloid precursors, ring sideroblasts Not done Alive (4 years 9 months)
21
6 Failure to thrive 4 Difficulty feeding, lethargy
MRI Status (age)
Growth Speech BM aspirate
21 Anemia thrombocytopenia Pancreatic insufficiency (17) Not involved Liver failure and cholestasis (20) Tubulopathy (17) Mild hypotonia (18) Yes Yes Recurrent acute pancreatitis, pancreatic cyst, 3-OH isobutyric aciduria Failure to thrive Normal Vacuolated erythroid/myeloid precursors, no ring sideroblasts Performed, Normal Deceased (23 months) Age at diagnosis (months) Bone marrow Exocrine pancreas (months) Endocrine pancreas (months) Liver (months) Kidney (months) Nervous system (months) Recurrent infections Transfusion Peculiar features
Onset (months) First medical issues
2 Failure to thrive
6 Failure to thrive, partial seizures 45 Pancytopenia Pancreatic insufficiency (44) Not involved Acute hepatitis focal fibrosis steatosis (30) Tubulopathy (8) Seizure (8–12) Hypotonia (45) Tremor (48) Yes Yes Fanconi syndrome symptomatic, hypocalcemia Failure to thrive Delayed Hypoplasia, macrocytosis, anisocytosis, and decrease iron storage Performed, Abnormal Alive (6 years)
Patient 4
4.5
623
Patient 3 Patient 2 Patient 1
TABLE I. Clinical Features of Patients with Pearson Syndrome
Average (months)
CRIPPA ET AL.
organic acids showed massive lactic (> 20,000 mmol/mol creatinine, normal 0–150 mmol/mol creatinine), and pyruvic aciduria (1,255 mmol/mol creatinine, normal 0–30 mmol/mol creatinine) along with increased excretion of intermediates of the citric cycle, severe ketonuria, and branched chain ketoaciduria. He also had increased serum lactate (3–3.9 mmol/L, normal 0.7–2.1 mmol/L) and lactate/pyruvate ratio (27). There was mild macrocytic anemia (hemoglobin 9.2 g/dl, normal 11.5–13.5 g/dl; mean corpuscular volume 89.2 fL, normal 75–87 fl). The biochemical profile and the clinical presentation were, at this point, highly suggestive of a mitochondrial disorder. Comprehensive mtDNA analysis showed a large heteroplasmic mtDNA deletion consistent with Pearson syndrome. He was found to have exocrine pancreatic insufficiency and was placed on pancreatic enzymes supplementation. He stopped eating by mouth and began to have more frequent emesis even with exclusive G-tube feeds. At 4 years, he began to have hand and head tremors and became unable to walk without assistance due to progressive weakness. Brain MRI revealed multiple focal areas of abnormal T2 signal and restricted diffusion within the subcortical white matter of both cerebral hemispheres consistent with acute infarcts. These abnormal imaging findings progressed over time in concert with his worsening neurological picture.
Patient 3 A 6-month-old female presented non-responsive with severe anemia (hemoglobin 2.5 g/dl, normal 11.5–13.5 g/dL), thrombocytopenia (platelets count 73 k/ml, normal 150–400 k/ml) and leukopenia (5 k/ml, normal 6–17 k/ml), mildly decreased lymphocytes (3.4 k/mL, normal 4–10 k/mL), and decreased neutrophils (1.4 k/ml, normal 1.5–8.5 k/ml). Lactate was elevated (7 mmol/L, normal 0.7–2.1 mmol/L). She required transfusions of packed red blood cells every 2 to 3 weeks and weekly platelet transfusions. Her initial bone marrow was normal, but at 10 months, there was hypocellular marrow with vacuolated erythroid and myeloid precursors and increased ring sideroblasts. These features suggested Pearson syndrome. She had feeding problems and chronic emesis along with abdominal pain. She required multiple hospitalizations for infections and transfusions. Mitochondrial deletion testing performed at 11 months of age showed a deletion of approximately 4.6 kb in 60–70% of her peripheral blood cells. She was started on carnitine, ubiquinone, vitamins, and iron chelation therapy. At 2.5 years, her absolute neutrophil count normalized, followed by normalization of other blood cell lineages. Her exocrine pancreatic function was normal by fecal fat studies, but she had hyperglycemia and was diagnosed with diabetes mellitus type 1 requiring insulin (3 units per day). She had mild elevation of liver enzymes (ALT 87 U/L, normal 5–45 U/L; AST 94 U/L, normal 20–60 U/L) and cholelithiasis without biliary ductal dilatation. Her development was appropriate for age and her physical exam was remarkable for hepatomegaly and mildly reduced muscle tone. Urine organic acids had severe lactic (7,602 mmol/mol creatinine, normal 0–150 mmol/mol creatinine) and pyruvic aciduria (400 mmol/mol creatinine, normal 0–30 mmol/mol creatinine) with ketonuria, branched-chain keto aciduria, and increased excretion of para-hydroxy phenyl organic
-2
B
300
20
200
10
100
0
-4
0 0
1
2
3
0 -2 -4
C
8
3
4
5
D
40
6
30
4
20
2
10 0 0
1
2
3
4
5 0
1
2
3
4
5
Age, years
2 0 -2 -4 6
2
0
C
0
1
RDW, %
RBC, 1000/ μL
2
4
5 0
Age, years
B
4
4
Platelets, 1000/ μL
0
A
30
12 18 24 30 36 42 48 54
AGE, Months
E
15
100 10 50
5 0
0 0
FIG. 1. Growth parameters in patients with Pearson syndrome. Growth is reported as standard deviations above (positive numbers) or below (negative numbers) average (set at 0 on the Y axis). Symbols represent different patients.
acids. Urine amino acids showed severe generalized amino aciduria, indicating renal tubular dysfunction. Plasma amino acids had marked elevation of alanine (1,082 mM/L, normal 240–600 mM/L), suggesting lactic acidosis and undetectable citrulline. Her urinalysis showed significant glucosuria and traces of hemoglobin. She was started on ubiquinol and citrulline supplementation. At 3 years of age, blood counts stabilized without transfusions. She developed pancreatic exocrine dysfunction and she was placed on pancreatic enzymes. She continues to be stable at 3.5 years of age. Her growth, speech, and motor development are on target.
Patient 4 A 6-month-old female presented with respiratory symptoms suggestive of pneumonia. Her physical exam was remarkable for deceleration of weight gain from the 10th to 3rd centile, irritability, skin pallor, lung crackles, and mild hepatomegaly. She was positive for influenza A. She was found to have pancytopenia with macrocytic anemia: low white blood count (3 K/ml, normal 6–17 k/ml), low hemoglobin (5.3 g/dl, normal 11.5–13.5 g/dl),
F MCV, fL
2
Patient 1 Patient 2 Patient 3 Patient 4
Hemoglobin, g/dL
HEAD CIRCUMFERENCE HEIGHT (SD from mean) (SD from mean)
A
4
WBC, 1000/ μL
AMERICAN JOURNAL OF MEDICAL GENETICS PART A
WEIGHT (SD from mean)
624
1
2
3
4
5 0
1
2
3
4
5
Age, years FIG. 2. Hematological parameters in patients with Pearson syndrome. Note progressive normalization of values in children who survive past 2 years of age.
elevated mean corpuscular volume (88.9 fl, normal 70–86 fl), and low platelet count (52K/ml, normal 150–400 k/ml). A chest X-ray performed at that time showed cardiomegaly, most likely due to severe anemia. A bone marrow biopsy showed ring sideroblasts and cytoplasmic vacuoles in erythroid and myeloid precursors. There was mild elevation of liver enzymes (ALT 46, normal 5–45 U/L; AST 107, normal 20–60 U/L) and lipase (224 U/l, normal 100–150 U/L). Plasma amino acids showed markedly elevated alanine (1,559 mM, normal 240–600 mM) low citrulline (2 mM, normal 10–60 mM/L), and low ornithine (19 mM/L, normal 20–135 mM/L). Urine organic acids showed mild lactic and pyruvic aciduria along with increased lactate/ pyruvate ratio (17.5). She had an increased excretion of intermediates of the citric cycle, dicarboxylic aciduria, and mild ketonuria. A heteroplasmic 5 kb mtDNA deletion was identified in peripheral cells. She received packed RBC transfusions and both anemia and cardiomegaly improved. She was placed on pancreatic enzymes. At 2 years of age, she no longer requires transfusions, and has normal growth and development.
CRIPPA ET AL.
625
FIG. 3. Pancreatic and liver pathology in Pearson Syndrome. (A) Pancreas. The pancreatic tissue showed preservation of islets and ductular structures, with fibrosis and loss of acini. Focal acute, neutrophilic inflammation within ducts was also noted. (B) Liver. The trichrome stain highlights the variable fibrosis and patchy cirrhosis seen in the liver. (C–E) Liver. Frequent pericentral loose fibrosis around terminal hepatic venules, as well as bridging fibrosis involving both portal and centrilobular zones (C). The liver showed patchy fibrosis and cirrhosis, with variable hepatocyte binucleation (consistent with regeneration), swelling, and vacuolation (D) There was focally prominent canalicular and intrahepatocytic cholestasis, as well as patchy exhuberant bile ductular proliferation with focal bile plugging (E).
RESULTS AND DISCUSSION TABLE II. Plasma Amino Acids in Patients with Pearson Syndrome Aminoacid Alanine Arginine Aspartic Acid Citrulline Cystine Glutamate Glutamine Glycine Histidine Leucine Lysine Methionine Ornithine Phenylalanine Proline Serine Taurine Threonine Tyrosine Valine
Average
SD
Normal ranges (mmol/L)
730.8 39.1 7.3 23.7 16.7 73.3 544.3 263.4 99.8 99.7 105.6 29.5 34.6 47.5 317.8 170.6 46.2 126.6 43.7 180.0
398.7 29.1 4.3 56.2 5.6 37.7 229.8 85.0 45.5 93.7 79.4 29.8 27.9 22.6 157.6 69.3 21.1 79.2 48.5 145.8
240–600 40–160 0–20 10–60 7–70 10–120 410–700 140–490 50–130 60–230 80–250 17–53 20–135 30–80 110–500 60–200 25–80 60–220 30–120 140–350
Values represent averages with the SD indicated. Averages outside the normal range are indicated by underlining.
Table I summarizes the clinical features of our patients with Pearson syndrome. Three of four patients presented with failure to thrive between 6 and 9 months of age (Fig. 1) and subsequently developed other symptoms that prompted to the correct diagnosis. Figure 1 shows the growth parameters in all patients. Short stature became evident over time in the surviving patients, whereas head circumference (and in some cases psychomotor development) were relatively unaffected. All patients had evidence of bone marrow involvement, with two of four patients having severe pancytopenia at presentation and all eventually developed bone marrow involvement. All cell lineages were involved (Fig. 2), with platelets being the most severely affected. There was macrocytosis with an increase in red cell distribution width (RDW) (anisocytosis). In the patient who died at about 2 years of age, white blood cells increased in response to an infection, but platelets (and red blood cells) remained suppressed. In patients who survived past 2 years of age, there was a progressive decrease in the need for transfusions until bone marrow sufficiency was achieved. There was a normalization of cell counts and all parameters, with a persistence of mild macrocytosis (Fig. 2F). By contrast, exocrine pancreatic insufficiency developed in all patients between 7 and 44 months of age. This was persistent over
A
B R2=0.09 p=0.19
100 50
100 50
2
R =0.45 p
Biochemical Abnormalities in Pearson Syndrome Beatrice Letizia Crippa,1,2 Eyby Leon,1 Amy Calhoun,1,6 Amy Lowichik,3,4 Marzia Pasquali,1,3,5 and Nicola Longo1,3,5* 1
Department of Pediatrics, Division of Medical Genetics, University of Utah, Salt Lake City, Utah
2
University of Milano, Milan, Italy Department of Pathology, University of Utah, Salt Lake City, Utah
3 4
Primary Children’s Hospital, Salt Lake City, Utah
5
ARUP Laboratories, University of Utah, Salt Lake City, Utah Department of Pediatrics, Division of Genetics and Metabolism, University of Minnesota, Minneapolis, Minnesota
6
Manuscript Received: 30 September 2014; Manuscript Accepted: 15 December 2014
Pearson marrow-pancreas syndrome is a multisystem mitochondrial disorder characterized by bone marrow failure and pancreatic insufficiency. Children who survive the severe bone marrow dysfunction in childhood develop Kearns–Sayre syndrome later in life. Here we report on four new cases with this condition and define their biochemical abnormalities. Three out of four patients presented with failure to thrive, with most of them having normal development and head size. All patients had evidence of bone marrow involvement that spontaneously improved in three out of four patients. Unique findings in our patients were acute pancreatitis (one out of four), renal Fanconi syndrome (present in all patients, but symptomatic only in one), and an unusual organic aciduria with 3-hydroxyisobutyric aciduria in one patient. Biochemical analysis indicated low levels of plasma citrulline and arginine, despite low-normal ammonia levels. Regression analysis indicated a significant correlation between each intermediate of the urea cycle and the next, except between ornithine and citrulline. This suggested that the reaction catalyzed by ornithine transcarbamylase (that converts ornithine to citrulline) might not be very efficient in patients with Pearson syndrome. In view of low-normal ammonia levels, we hypothesize that ammonia and carbamylphosphate could be diverted from the urea cycle to the synthesis of nucleotides in patients with Pearson syndrome and possibly other mitochondrial disorders. Ó 2015 Wiley Periodicals, Inc.
Key words: Pearson syndrome; mitochondrial disease; organic aciduria; orotic acid; plasma amino acids; uridine
INTRODUCTION Pearson marrow-pancreas syndrome was first described in 1979 as a fatal multisystem mitochondrial disorder that occurs in early infancy [Pearson et al., 1979; DiMauro and Hirano, 1993]. Bone marrow failure is the first typical feature of Pearson syndrome with isolated macrocytic sideroblastic anemia, frequently transfusion-
Ó 2015 Wiley Periodicals, Inc.
How to Cite this Article: Crippa BL, Leon E, Calhoun A, Lowichik A, Pasquali M, Longo N. 2015. Biochemical abnormalities in Pearson syndrome. Am J Med Genet Part A 167A:621–628.
dependent, or in association with thrombocytopenia and neutropenia [Pearson et al., 1979]. The presence of vacuolization in granulous and erythroblastic progenitors, visible on the myelogram, and the presence of ringed sideroblasts are typical feature of Pearson syndrome [Muraki et al., 1997; Pearson et al., 1979]. Dysfunction of the exocrine pancreas due to fibrosis is also present in Pearson syndrome with chronic diarrhea, malabsorption, and failure to thrive [Pearson et al., 1979]. Children who survive the severe bone marrow dysfunction develop Kearns–Sayre syndrome later in life (usually before 20 years of age) [McShane et al., 1991; Muraki et al., 1997]. There is no effective therapy for this condition. Other affected organ systems include the kidney (tubulopathy and aminoaciduria) [Superti-Furga et al., 1993; Atale et al., 2009], liver (hepatomegaly, cytolysis, and cholestasis) [Superti-Furga et al., 1993; Atale et al., 2009], endocrine glands (diabetes mellitus, adrenal insufficiency) [Williams et al., 2012], neuromuscular system, and heart [Krauch et al., 2002]. Identification of mitochondrial DNA rearrangements with a spectrum of mtDNA deletions confirms the diagnosis [Rotig et al., 1995] Pearson syndrome can be difficult to distinguish from other mitochondrial disorders when multiple organs are involved early, Conflict of interest: none. Correspondence to: Nicola Longo, M.D., Ph.D., Division of Medical Genetics, Department of Pediatrics, University of Utah, 2C412 SOM, 50 North Mario Capecchi Drive, Salt Lake City, UT 84132. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): DOI 10.1002/ajmg.a.36939
621
622 but in many cases the pancreatic involvement can reveal the diagnosis [Fellman and Kotarsky, 2011; Honzik et al., 2012]. Patients with Pearson syndrome can have lactic acidemia and abnormal urine organic acids, with methylglutaconic aciduria and increased intermediates of the citric acid cycle [Gibson et al., 1992; Ribes et al., 1993; Superti-Furga et al., 1993]. Alterations in plasma amino acids have also been reported [Ribes et al., 1993], but no extensive studies have been performed in these patients. Here we report on four new patients with Pearson syndrome, some with unique clinical manifestations. Some of these patients had unusual biochemical profiles that might aid in their diagnosis, have utility for clinical monitoring, and lead to novel therapies.
MATERIALS AND METHODS A retrospective chart review of the clinical course and laboratory test results was performed for patients followed in our clinic for Pearson syndrome. Informed consent was provided by patients for DNA testing. Testing for common mtDNA point mutations and the common Pearson syndrome deletion were performed in peripheral blood using standard PCR amplification and restriction enzyme digestion at Baylor College of Medical Genetics Laboratory (Houston, TX). Urine organic acids were analyzed by gas chromatography/mass spectrometry; plasma amino acids by ion exchange chromatography; and plasma acylcarnitine profile by tandem mass spectrometry using standard procedures at ARUP Laboratories, Salt Lake City, Utah. Routine testing was performed in a pediatric hospital using standard procedures and age appropriate reference ranges.
AMERICAN JOURNAL OF MEDICAL GENETICS PART A she became transfusion dependent for both red blood cells and platelets. She developed progressive liver failure with cholestasis with severe intestinal dysfunction at 20 months of age requiring total parenteral nutrition. She died at 23 months of age due to septic shock with multiorgan failure. Laboratory studies collected from her initial presentation at 9 month of age to her death at 23 months indicated chronic l actic acidosis (lactic acid 10 3.8 mmol/L, n ¼ 55, normal 0.7–2.1 mmol/L) and abnormal plasma amino acids with elevated alanine (676 386 mM, range 156–1503, n ¼ 18, normal 240–600 mM), low arginine (38 32 mM, range 11–121, n ¼ 18, normal 40–160 mM), low citrulline (despite some values were collected while the patient was on supplements, 28 63 mM, range 0–275, n ¼ 18, normal 10–60 mM), and low ornithine (33 29 mM, range 9–98, n ¼ 18, normal 20–135 mM). Amino acid values fluctuated depending on citrulline supplementation (200 mg/ kg per day starting at 17 months of life). Ammonia levels were nearly always low, with a single value in the normal range (15.6 13.5 mM, n ¼ 5, normal 21–50 mM). Urine organic acids showed a persistent excretion of lactic, pyruvic, 3-hydroxyisobutyric, and 2-ethyl-3-hydroxy propionic acids. These findings suggested mitochondrial dysfunction. Mitochondrial DNA testing revealed heteroplasmy for a large deletion of mtDNA and diagnosis of Pearson Syndrome was made at 21 months. Bone marrow biopsy at 21 months of age indicated hypoplastic marrow with vacuolated erythroid cells that in the presence of megaloblastic changes were supportive of the diagnosis of Pearson syndrome, despite lack of ring sideroblasts.
Patient 2 CLINICAL REPORT Patient 1 A previously healthy female was diagnosed with failure to thrive at 9 months of age, with weight reaching the 1st centile. At 13 months, she was admitted to the hospital for dehydration due to viral gastroenteritis that required admission to the intensive care unit. She had mild metabolic acidosis (anion gap 9 mmol/L, normal 3–16 mmol/L; CO2 13 mmol/L, normal 18–24 mmol/L) and elevated lactate level (4.4 mmol/L; normal 0.7–2.1 mmol/L). Blood counts were normal except for mild thrombocytopenia (platelets 139, normal 150–400 k/ml) and macrocytosis (mean corpuscular volume 89.2 fl, normal 70–86 fl). She had persistent vomiting and abdominal pain with a cyst in the pancreatic tail, normal appearance of the pancreas, and elevated lipase (1,639 U/L, normal 100–150 U/L) prompting a diagnosis of acute pancreatitis. During admission, she developed pancytopenia that improved spontaneously and was initially attributed to viral suppression. She had recurrent pancreatitis 3.5 months later with a peak lipase of 2,662 U/L and then four more times (peak lipase 2,908 U/L) with no major alterations of her blood counts. She was found to have exocrine pancreatic failure at 17 months. Renal tubulopathy was evidenced by persistent aminoaciduria, glycosuria, and tubular acidosis. At 18 months of age, she developed hypotonia, but her brain MRI was normal with normal spectroscopy. Her speech development was normal. Anemia progressively worsened and
A 6-month-old male presented with partial seizures and failure to thrive. He had multiple hospitalizations starting at 8 months of age for renal tubular acidosis, symptomatic hypocalcemia with tetanus, failure to thrive, recurrent infections, dehydration, and pancytopenia requiring blood transfusions. Laboratory studies indicated mildly elevated liver enzymes, alkaline phosphatase, and aldolase (12.2 U/L, normal 3.4–11.6 U/L), with normal urine amino acids (at an outside laboratory) and ammonia levels. He had multiple biopsies, including muscle that showed increased connective tissue suggestive of myopathy; his liver showed mild acute hepatitis with focal fibrosis and patchy steatosis; and his bone marrow was hypoplastic with macrocytosis, anisocytosis, and decreased iron storage. He had mild motor and speech delays. At 2.5 years of age, his height and weight were below the 3rd centile. He was talking in sentences and his muscle tone was mildly reduced. He was receiving sodium citrate, carnitine, calcium carbonate, and iron. At 3 years of age, he began to have daily emesis and diarrhea. He also developed multiple dental abscesses requiring surgical drainage. He was hospitalized twice and found to have severe lactic acidosis with normal anion gap. At 3 years 8 months, urine amino acids showed severe generalized aminoaciduria, proteinuria (protein/creatinine ratio 2.2, normal 0–0.2 mg/mg creatinine; albumin/creatinine ratio 707–720, normal 10–150 mg/g creatinine), suggesting impaired renal tubular function. Plasma amino acids showed mildly elevation of alanine (658 mM/L, normal 240–600 mM), low arginine (30 mM, normal 40–160 mM), normal citrulline (15 mM); urine
26.2 30 20.7 15.2 26.1
6 Pancytopenia Pancreatic insufficiency (8) Not involved Liver dysfunction (6) Tubulopathy (6) Not affected Yes Yes Simultaneous involvement of bone marrow, exocrine pancreas, liver and kidney Failure to thrive Normal Vacuolated erythroid/myeloid precursors, ring sideroblasts Not done Alive (2 years 8 months) 12 Anemia, thrombocytopenia, neutropenia Pancreatic insufficiency (36) DM type 1 (30) Liver dysfunction (24), cholelithiasis (30) Tubulopathy (30) Not affected No Yes DM type 1, late-onset pancreatic exocrine dysfunction Failure to thrive Normal Vacuolated erythroid/myeloid precursors, ring sideroblasts Not done Alive (4 years 9 months)
21
6 Failure to thrive 4 Difficulty feeding, lethargy
MRI Status (age)
Growth Speech BM aspirate
21 Anemia thrombocytopenia Pancreatic insufficiency (17) Not involved Liver failure and cholestasis (20) Tubulopathy (17) Mild hypotonia (18) Yes Yes Recurrent acute pancreatitis, pancreatic cyst, 3-OH isobutyric aciduria Failure to thrive Normal Vacuolated erythroid/myeloid precursors, no ring sideroblasts Performed, Normal Deceased (23 months) Age at diagnosis (months) Bone marrow Exocrine pancreas (months) Endocrine pancreas (months) Liver (months) Kidney (months) Nervous system (months) Recurrent infections Transfusion Peculiar features
Onset (months) First medical issues
2 Failure to thrive
6 Failure to thrive, partial seizures 45 Pancytopenia Pancreatic insufficiency (44) Not involved Acute hepatitis focal fibrosis steatosis (30) Tubulopathy (8) Seizure (8–12) Hypotonia (45) Tremor (48) Yes Yes Fanconi syndrome symptomatic, hypocalcemia Failure to thrive Delayed Hypoplasia, macrocytosis, anisocytosis, and decrease iron storage Performed, Abnormal Alive (6 years)
Patient 4
4.5
623
Patient 3 Patient 2 Patient 1
TABLE I. Clinical Features of Patients with Pearson Syndrome
Average (months)
CRIPPA ET AL.
organic acids showed massive lactic (> 20,000 mmol/mol creatinine, normal 0–150 mmol/mol creatinine), and pyruvic aciduria (1,255 mmol/mol creatinine, normal 0–30 mmol/mol creatinine) along with increased excretion of intermediates of the citric cycle, severe ketonuria, and branched chain ketoaciduria. He also had increased serum lactate (3–3.9 mmol/L, normal 0.7–2.1 mmol/L) and lactate/pyruvate ratio (27). There was mild macrocytic anemia (hemoglobin 9.2 g/dl, normal 11.5–13.5 g/dl; mean corpuscular volume 89.2 fL, normal 75–87 fl). The biochemical profile and the clinical presentation were, at this point, highly suggestive of a mitochondrial disorder. Comprehensive mtDNA analysis showed a large heteroplasmic mtDNA deletion consistent with Pearson syndrome. He was found to have exocrine pancreatic insufficiency and was placed on pancreatic enzymes supplementation. He stopped eating by mouth and began to have more frequent emesis even with exclusive G-tube feeds. At 4 years, he began to have hand and head tremors and became unable to walk without assistance due to progressive weakness. Brain MRI revealed multiple focal areas of abnormal T2 signal and restricted diffusion within the subcortical white matter of both cerebral hemispheres consistent with acute infarcts. These abnormal imaging findings progressed over time in concert with his worsening neurological picture.
Patient 3 A 6-month-old female presented non-responsive with severe anemia (hemoglobin 2.5 g/dl, normal 11.5–13.5 g/dL), thrombocytopenia (platelets count 73 k/ml, normal 150–400 k/ml) and leukopenia (5 k/ml, normal 6–17 k/ml), mildly decreased lymphocytes (3.4 k/mL, normal 4–10 k/mL), and decreased neutrophils (1.4 k/ml, normal 1.5–8.5 k/ml). Lactate was elevated (7 mmol/L, normal 0.7–2.1 mmol/L). She required transfusions of packed red blood cells every 2 to 3 weeks and weekly platelet transfusions. Her initial bone marrow was normal, but at 10 months, there was hypocellular marrow with vacuolated erythroid and myeloid precursors and increased ring sideroblasts. These features suggested Pearson syndrome. She had feeding problems and chronic emesis along with abdominal pain. She required multiple hospitalizations for infections and transfusions. Mitochondrial deletion testing performed at 11 months of age showed a deletion of approximately 4.6 kb in 60–70% of her peripheral blood cells. She was started on carnitine, ubiquinone, vitamins, and iron chelation therapy. At 2.5 years, her absolute neutrophil count normalized, followed by normalization of other blood cell lineages. Her exocrine pancreatic function was normal by fecal fat studies, but she had hyperglycemia and was diagnosed with diabetes mellitus type 1 requiring insulin (3 units per day). She had mild elevation of liver enzymes (ALT 87 U/L, normal 5–45 U/L; AST 94 U/L, normal 20–60 U/L) and cholelithiasis without biliary ductal dilatation. Her development was appropriate for age and her physical exam was remarkable for hepatomegaly and mildly reduced muscle tone. Urine organic acids had severe lactic (7,602 mmol/mol creatinine, normal 0–150 mmol/mol creatinine) and pyruvic aciduria (400 mmol/mol creatinine, normal 0–30 mmol/mol creatinine) with ketonuria, branched-chain keto aciduria, and increased excretion of para-hydroxy phenyl organic
-2
B
300
20
200
10
100
0
-4
0 0
1
2
3
0 -2 -4
C
8
3
4
5
D
40
6
30
4
20
2
10 0 0
1
2
3
4
5 0
1
2
3
4
5
Age, years
2 0 -2 -4 6
2
0
C
0
1
RDW, %
RBC, 1000/ μL
2
4
5 0
Age, years
B
4
4
Platelets, 1000/ μL
0
A
30
12 18 24 30 36 42 48 54
AGE, Months
E
15
100 10 50
5 0
0 0
FIG. 1. Growth parameters in patients with Pearson syndrome. Growth is reported as standard deviations above (positive numbers) or below (negative numbers) average (set at 0 on the Y axis). Symbols represent different patients.
acids. Urine amino acids showed severe generalized amino aciduria, indicating renal tubular dysfunction. Plasma amino acids had marked elevation of alanine (1,082 mM/L, normal 240–600 mM/L), suggesting lactic acidosis and undetectable citrulline. Her urinalysis showed significant glucosuria and traces of hemoglobin. She was started on ubiquinol and citrulline supplementation. At 3 years of age, blood counts stabilized without transfusions. She developed pancreatic exocrine dysfunction and she was placed on pancreatic enzymes. She continues to be stable at 3.5 years of age. Her growth, speech, and motor development are on target.
Patient 4 A 6-month-old female presented with respiratory symptoms suggestive of pneumonia. Her physical exam was remarkable for deceleration of weight gain from the 10th to 3rd centile, irritability, skin pallor, lung crackles, and mild hepatomegaly. She was positive for influenza A. She was found to have pancytopenia with macrocytic anemia: low white blood count (3 K/ml, normal 6–17 k/ml), low hemoglobin (5.3 g/dl, normal 11.5–13.5 g/dl),
F MCV, fL
2
Patient 1 Patient 2 Patient 3 Patient 4
Hemoglobin, g/dL
HEAD CIRCUMFERENCE HEIGHT (SD from mean) (SD from mean)
A
4
WBC, 1000/ μL
AMERICAN JOURNAL OF MEDICAL GENETICS PART A
WEIGHT (SD from mean)
624
1
2
3
4
5 0
1
2
3
4
5
Age, years FIG. 2. Hematological parameters in patients with Pearson syndrome. Note progressive normalization of values in children who survive past 2 years of age.
elevated mean corpuscular volume (88.9 fl, normal 70–86 fl), and low platelet count (52K/ml, normal 150–400 k/ml). A chest X-ray performed at that time showed cardiomegaly, most likely due to severe anemia. A bone marrow biopsy showed ring sideroblasts and cytoplasmic vacuoles in erythroid and myeloid precursors. There was mild elevation of liver enzymes (ALT 46, normal 5–45 U/L; AST 107, normal 20–60 U/L) and lipase (224 U/l, normal 100–150 U/L). Plasma amino acids showed markedly elevated alanine (1,559 mM, normal 240–600 mM) low citrulline (2 mM, normal 10–60 mM/L), and low ornithine (19 mM/L, normal 20–135 mM/L). Urine organic acids showed mild lactic and pyruvic aciduria along with increased lactate/ pyruvate ratio (17.5). She had an increased excretion of intermediates of the citric cycle, dicarboxylic aciduria, and mild ketonuria. A heteroplasmic 5 kb mtDNA deletion was identified in peripheral cells. She received packed RBC transfusions and both anemia and cardiomegaly improved. She was placed on pancreatic enzymes. At 2 years of age, she no longer requires transfusions, and has normal growth and development.
CRIPPA ET AL.
625
FIG. 3. Pancreatic and liver pathology in Pearson Syndrome. (A) Pancreas. The pancreatic tissue showed preservation of islets and ductular structures, with fibrosis and loss of acini. Focal acute, neutrophilic inflammation within ducts was also noted. (B) Liver. The trichrome stain highlights the variable fibrosis and patchy cirrhosis seen in the liver. (C–E) Liver. Frequent pericentral loose fibrosis around terminal hepatic venules, as well as bridging fibrosis involving both portal and centrilobular zones (C). The liver showed patchy fibrosis and cirrhosis, with variable hepatocyte binucleation (consistent with regeneration), swelling, and vacuolation (D) There was focally prominent canalicular and intrahepatocytic cholestasis, as well as patchy exhuberant bile ductular proliferation with focal bile plugging (E).
RESULTS AND DISCUSSION TABLE II. Plasma Amino Acids in Patients with Pearson Syndrome Aminoacid Alanine Arginine Aspartic Acid Citrulline Cystine Glutamate Glutamine Glycine Histidine Leucine Lysine Methionine Ornithine Phenylalanine Proline Serine Taurine Threonine Tyrosine Valine
Average
SD
Normal ranges (mmol/L)
730.8 39.1 7.3 23.7 16.7 73.3 544.3 263.4 99.8 99.7 105.6 29.5 34.6 47.5 317.8 170.6 46.2 126.6 43.7 180.0
398.7 29.1 4.3 56.2 5.6 37.7 229.8 85.0 45.5 93.7 79.4 29.8 27.9 22.6 157.6 69.3 21.1 79.2 48.5 145.8
240–600 40–160 0–20 10–60 7–70 10–120 410–700 140–490 50–130 60–230 80–250 17–53 20–135 30–80 110–500 60–200 25–80 60–220 30–120 140–350
Values represent averages with the SD indicated. Averages outside the normal range are indicated by underlining.
Table I summarizes the clinical features of our patients with Pearson syndrome. Three of four patients presented with failure to thrive between 6 and 9 months of age (Fig. 1) and subsequently developed other symptoms that prompted to the correct diagnosis. Figure 1 shows the growth parameters in all patients. Short stature became evident over time in the surviving patients, whereas head circumference (and in some cases psychomotor development) were relatively unaffected. All patients had evidence of bone marrow involvement, with two of four patients having severe pancytopenia at presentation and all eventually developed bone marrow involvement. All cell lineages were involved (Fig. 2), with platelets being the most severely affected. There was macrocytosis with an increase in red cell distribution width (RDW) (anisocytosis). In the patient who died at about 2 years of age, white blood cells increased in response to an infection, but platelets (and red blood cells) remained suppressed. In patients who survived past 2 years of age, there was a progressive decrease in the need for transfusions until bone marrow sufficiency was achieved. There was a normalization of cell counts and all parameters, with a persistence of mild macrocytosis (Fig. 2F). By contrast, exocrine pancreatic insufficiency developed in all patients between 7 and 44 months of age. This was persistent over
A
B R2=0.09 p=0.19
100 50
100 50
2
R =0.45 p
