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| Web: | mayocliniclabs.com |
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| Email: | mcl@mayo.edu |
| Telephone: | 800-533-1710 |
| International: | +1 855-379-3115 |
| Values are valid only on day of printing | |
A 31-month-old girl presented with cutaneous ulcerations on her face, hands, and arms. She has hepatosplenomegaly and slight elevations in liver function tests. The care team suspects porphyria and orders biochemical and molecular testing. Blood, urine, and fecal samples were tested for any abnormal accumulation of porphyrins and intermediates of the porphyrin biosynthesis pathway. Erythrocyte uroporphyrinogen decarboxylase (UROD) activity was markedly reduced by 95% when compared to normal individuals. Urine and fecal porphyrin analysis detected abnormally high excretion of uroporphyrinogen III (Figure 1). The excretion of Coproporphyrinogen III precursors (heptacarboxyl porphyrin, hexacarboxylporphyrin, and pentacarboxyporphyrin) were markedly elevated in urine (Figure 1A; this test does not separate isomer I to isomer III species). In addition, the excretion of isocoproporphyrins was detected in feces (Figure 1B). The excretions of aminolevulinic acid and porphobilinogen were normal. Fractionation of protoporphyrins in whole blood showed an elevation of zinc-associated protoporphyrins (Figure 2). Molecular testing using a 10-gene porphyria panel identified two pathogenic variants in UROD.
The correct answer is ...
Hepatoerythropoietic porphyria.
Hepatoerythropoietic porphyria (HEP) is an extremely rare, autosomal recessive genetic disorder characterized by deficiency of the enzyme UROD. Fewer than 100 cases have been reported. Most affected individuals have a profound deficiency of this enzyme, and the onset of the disorder is usually during infancy or early childhood. Symptoms and severity can vary, but patients generally experience severe cutaneous photosensitivity and fragile skin. Affected skin can become discolored and prone to bacterial infection. Hypertrichosis on sun-exposed skin and erythrodontia are common. Bone fragility and hemolytic anemia often result. Abnormalities in liver function may also occur.1
Pathogenic variants in UROD can cause two porphyria conditions: familial porphyria cutanea tarda (PCT type II) and hepatoerythropoietic porphyria (HEP).2 A significant biochemical and clinical overlap exists between the two conditions. Reduced UROD activity in all tissues leads to the accumulation of uroporphyrinogen III and precursors of coproporphyrinogen III synthesis (heptacarboxylporphyrin, hexacarboxylporphyrin, and pentacarboxyporphyrin).
Although the UROD activity is low in both PCT type II and HEP, a UROD activity of <10% is typically seen in HEP patients.3 This contrasts with PCT type II patients who have UROD activity of approximately 50%. Secondly, PCT is usually a late-onset disease with symptoms first manifesting in the early 30s. The patient presented here has an early onset of disease, which is characteristic of HEP. Additionally, PCT type II is inherited in an autosomal dominant manner while HEP is inherited as an autosomal recessive trait. Increased isocoproporphyrins in feces are characteristic of both PCT type II and HEP.4 In contrast to PCT, erythrocyte zinc-associated protoporphyrin is increased in HEP.
Porphyria cutanea tarda type II (vs hepatoerythropoietic porphyria)
Porphyria cutanea tarda is the most common porphyria. Although porphyria cutanea tarda is a late-onset condition, early-onset cases have been reported. Three types of PCT exist: type 1 (acquired or sporadic, 80%); PCT, type 2 (familial PCT, 20%); and type 3 (no pathogenic variants in UROD, cause unknown).5
Congenital erythropoietic porphyria
Congenital erythropoietic porphyria (CEP) presents in infancy and is the rarest porphyria. CEP is an autosomal recessive porphyria caused by pathogenic variants in the uroporphyrinogen III synthase (UROS). Patients present with cutaneous photosensitivity (redness, blistering, thickening) and increased vulnerability to skin infection. Erythrodontia is common. Vomiting, abdominal pain, and constipation may occur during attacks. Hepatic and hematological involvement has been reported.1 While CEP and HEP share some common clinical features, the biochemical phenotypes are different. Biochemically, uroporphyrinogen I and coproporphyrinogen I accumulate in the blood. Measurement of these analytes in fecal, blood, and urine samples can establish a diagnosis for CEP.
Erythropoietic protoporphyria
Erythropoietic protoporphyria (EPP) is caused by low ferrochelatase activity, the last enzyme in the heme biosynthetic pathway. While both EPP and HEP are classified as cutaneous porphyrias, the clinical and biochemical phenotypes are different. EPP patients have an immediate, painful reaction to sunlight while HEP manifests with severe, bullous sun sensitivity.6 Contrary to elevated zinc-associated protoporphyrin observed in HEP patients, EPP patients show excess accumulation of metal-free protoporphyrin in red blood cells, plasma, and fecal specimens. Additionally, the urine porphyrin profile is normal in EPP patients.
Follow-up
There is no cure for HEP. Avoidance of exposure to triggering agents and management of symptoms remains the key strategy for the improvement of patient outcomes.
Gerald Dayebgadoh, Ph.D.
Fellow, Clinical Biochemical Genetics
Mayo Clinic
Tricia Hall, Ph.D.
Senior Associate Consultant, Laboratory Genetics and Genomics
Mayo Clinic
Assistant Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science
A 58-year-old man underwent a chest X-ray after a minor elective surgery, and was found to have a 4 cm left pleural-based mass. His past medical history was significant for hypertension, hyperlipidemia, osteomalacia, and obesity. Surgical resection of the mass demonstrated loose arrays of spindled and stellate cells without nuclear atypia. The signing pathologist ordered in-situ hybridization of FGF23 mRNA, which showed overexpression.
The correct answer is ...
Osteomalacia.
This patient has a rare soft tissue neoplasm called a phosphaturic mesenchymal tumor. The neoplastic cells overexpress and secrete FGF23, a hormone that inhibits phosphate reabsorption by proximal renal tubules. Laboratory workup prior to resection may have shown hypophosphatemia, hyperphosphaturia, and normocalcemia. Many patients present with osteomalacia with recurrent bone fractures and vitamin D deficiency. The tumor-induced osteomalacia often resolves with resection of the tumor. Roughly half of these tumors possess an FN1-FGF1 gene fusion.
Peter Kundert, M.D., Ph.D.
Resident, Anatomic and Clinical Pathology
Mayo Clinic
Jorge Torres-Mora, M.D.
Consultant, Anatomic Pathology
Mayo Clinic
Assistant Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science
A 12-year-old girl presents to her primary care physician for evaluation of anxiety, panic symptoms, and low BMI. She denies intentional restrictive eating or binging/purging. At 18:34, the chemistry resident on-call is informed by the lab that the patient has a potassium value of 6.6 mmol/L. They are having difficulty reaching the primary care provider, and are wondering if this result could be due to contamination or some other type of laboratory error.
The correct answer is ...
New-onset Type I diabetes mellitus (T1DM).
Based on the potassium and glucose results, the patient was referred to the Emergency Department. Upon presentation to the ED, lab results are notable for sodium of 122 mmol/L, glucose of 896 mg/dL, lactate of 2.4 mmol/L, and beta-hydroxybutyrate of 1.6 mmol/L.
A diabetes diagnosis is confirmed by A1c of 18.0% and glucose of 896 mg/dL. At the time of presentation to the ED, the earlier hyperkalemia has resolved, but the patient is now hyponatremic.
New-onset T1DM, electrolyte abnormalities, and DKA/HHS
Are the observed electrolyte abnormalities consistent with the patient’s condition, or was some sort of laboratory or pre-analytical error involved? Significant electrolyte abnormalities can be associated with acute presentation of T1DM, especially in diabetic ketoacidosis (DKA) or hyperglycemic hyperosmolar state (HHS), which are often the first presentation in new-onset cases of Type I diabetes. Significant hyperkalemia is often seen in DKA/HHS,1,2 and can be attributed to multiple mechanisms, all of which contribute to a shift of potassium from the intracellular to extracellular compartment.
First, the absence of insulin, which activates the Na/K-ATPase at cell membranes and is therefore responsible for maintaining the intracellular potassium gradient, allows the loss of potassium from the intracellular to the extracellular space. Second, the presence of increased extra-cellular osmolarity due to marked hyperglycemia causes efflux of water from cells, accompanied by additional intracellular potassium. Finally, the presence of metabolic acidosis, as in DKA, is associated with extracellular shunting of potassium as hydrogen ions accumulate intracellularly, displacing positively charged potassium ions to balance intracellular and extracellular charge.
The hyponatremia observed upon presentation to the ED would also be consistent with marked hyperglycemia and/or DKA/HHS; increased extracellular osmolarity results in an expansion of the extracellular fluid volume, resulting in a dilutional hyponatremia.3 While pseudohyperkalemia by hemoconcentration due to the combination of fist-clenching and prolonged tourniquet time during phlebotomy is known to occur,4 this mechanism would not explain the other lab abnormalities seen in this case.
The diagnosis of T1DM was established by the presence of marked hyperglycemia, ketosis (urine ketones positive, beta-hydroxybutyrate elevated), and elevated A1c. The patient did not meet the criteria for DKA, not being frankly acidotic (venous pH 7.35) despite the presence of BOHB and an increased anion gap. The patient was also not classified as having HHS, having a calculated serum osmolality of 294 mOsm/kg (RI: 275-295); HHS is generally associated with osmolality in excess of 320 mOsm/kg. The patient was therefore admitted to pediatric general medicine and managed successfully with intravenous fluid administration and subcutaneous insulin. Prompt laboratory diagnosis and follow-up may have prevented a presentation of acute DKA, which can have significant associated morbidity and mortality.5
Additional testing for T1DM
The patient was also found to be positive for antibodies to islet antigen-2 (IA-2). While not necessary for the diagnosis of T1DM in most cases, autoantibodies against islet cell antigens including insulin, IA-2, glutamate decarboxylase 65 (GAD65) and zinc transporter ZnT8, can be helpful in determining an autoimmune etiology. Greater than 95% of patients with T1DM are positive for at least one of these auto-antibodies, and their presence can be helpful for distinguishing between Type 1 and Type 2 diabetes in ambiguous cases, such as late-onset diabetes, adolescent diabetes presenting without ketoacidosis, and adolescent onset diabetes in patients who are overweight or obese.6,7
Benjamin Andress, Ph.D.
Fellow, Clinical Chemistry
Mayo Clinic
Brad Karon, M.D., Ph.D.
Consultant, Clinical Core Laboratory Services
Mayo Clinic
Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science
An 81-year-old man presented with neutropenia and thrombocytopenia. Peripheral blood showed 9% blasts and bone marrow aspirate/biopsy showed 15% blasts (Figure 1) with basophilic cytoplasm, perinuclear clearing, and occasional cytoplasmic azurophilic granules. In the background was maturing granulopoiesis (>10% of total bone marrow cellularity). A subset of neutrophils were hypogranular and had Pelger Huet-like nuclei (Figure 2). Flow cytometry showed a blast population that was positive for CD34, CD45, CD117, HLA-DR, CD33(dim), and CD15(partial). Conventional cytogenetics revealed a t(8;21) (q22;q22.1) translocation in 8/20 metaphases (Figure 3).
The correct answer is ...
Acute myeloid leukemia with RUNX1::RUNX1T1.
As per the 5th edition of the World Health Organization Classification of Haematolymphoid Tumours, AML with RUNX1-RUNX1T1 is a defining genetic abnormality and is considered to be acute leukemia regardless of the blast count.1 The RUNX1-RUNX1T1 fusion was confirmed with a dual color double fusion probe set (Figure 4). The leukemic blasts in AML with RUNX1-RUNX1T1 exhibit a characteristic immunophenotype that includes expression of CD34, aberrant expression of the B-cell associated lymphoid markers CD19 and cytoplasmic CD79a, myeloid markers (CD13 and CD33) and CD56 is positive in some cases. Acute myeloid leukemia with maturation, NOS and acute myeloid leukemia without maturation, NOS are diagnoses of exclusion and do not fulfill criteria for any of the other categories of AML (e.g., recurrent genetic abnormality). Myelodysplastic neoplasm with increased blasts – 2 is a myeloid neoplasm with cytopenia(s) and dysplasia, lacks defining genetic abnormalities, and shows 5%-19% bone marrow blasts and/or 2%-19% peripheral blood blasts.
Arpan Samaddar, M.B.B.S.
Resident, Anatomic & Clinical Pathology
Mayo Clinic
Kaaren Reichard, M.D.
Consultant, Hematopathology
Mayo Clinic
Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science
A 26-year-old woman with a history of a childhood adrenal tumor and osteosarcoma was referred for genetic testing. A heterozygous pathogenic variant in the TP53 gene was identified (from blood), which is consistent with a diagnosis of Li Fraumeni syndrome (Figure 1). Subsequently, the patient elected to undergo IVF (two cycles) with preimplantation genetic testing, which did not identify the variant in any of 10 embryos. This prompted follow-up testing in additional tissues, including a new blood sample. Next-generation sequencing identified the variant in all her tested tissues at varying allele fractions (Figure 2).
The correct answer is ...
The mother is exhibiting mosaicism for the TP53 variant.
All 10 embryos inherited the wild-type TP53 allele.
Li Fraumeni syndrome (LFS) is an autosomal dominant cancer predisposition syndrome associated with pathogenic variants in the TP53 gene. The hallmark feature of LFS is development of multiple primary cancers with an early age of onset.1,2
The probability that all 10 embryos (from two cycles of IVF) inherited the wild-type allele for TP53 is less than 0.01% (1/2^10) and therefore highly unlikely for an autosomal dominant condition where approximately 50% of offspring would be expected to inherit the pathogenic allele.
The preimplantation genetic testing assay has poor sensitivity to detect the variant in embryonic cells.
The preimplantation genetic testing (PGT) assay determines the genotype of embryos for single-gene disorders before transfer to the uterus. It typically utilizes a few cells from the trophectoderm of the embryo at the blastocyst stage, which are cells that develop into the placenta.3 While misdiagnosis is possible due to false-positive or false-negative results, this may be observed in the context of aneuploidy (number of chromosomes is too many or too few) rather than in the context of a sequence alteration, as seen in this individual. Confirmation of PGT findings is always recommended by follow-up amniocentesis or chorionic villus sampling to address this possibility.
The TP53 variant is a result of clonal hematopoiesis of indeterminate potential (CHIP), and therefore is not heritable.
CHIP is defined as an age-related phenomenon occurring in healthy individuals (without overt malignancy) and is characterized by clonal expansion of hematopoietic stem cells or progenitor cells harboring hematologic malignancy-related genetic variants in specific genes, including TP53.4 They are more likely to be present at low allele fractions, as they are confined to subpopulations of hematologic cells. As these are somatically acquired variants, they will not be inherited by progeny and they will be absent from all other tissues, the latter being the differentiating feature between CHIP and inherited disorders such as LFS. Additionally, CHIP has been associated with numerous adverse conditions including increased risk of cardiac events, development of hematological malignancies, and poor overall survival in this context. Therefore, it is important to correctly elucidate the origin of this TP53 variant because clinical and reproductive-risk implications would be quite different in the context of CHIP versus LFS.
What is mosaicism?
During embryonic development, gastrulation results in the formation of three germ layers: endoderm, mesoderm, and ectoderm, each of which differentiates into various tissues of the body. When a new genetic alteration occurs in a zygote very early on, all daughter cells will carry that variant and it will be present in all germ layers/tissues tested. As a heterozygous variant, it will be detected at a VAF of approximately 50%. Additionally, it will be absent from the individual’s parents. This is termed a de novo variant. However, when a genetic alteration occurs at a later timepoint, it will affect a smaller percentage of cells and may not be present in all germ layers/ tissues. It may also be detected at a VAF <50%, depending on the time at which the variant was acquired, and the proportion of cells affected. This is termed mosaicism.5
In the laboratory, detection of a variant below a VAF of 30% raises the suspicion of mosaicism and is typically followed up by testing of a different tissue (helps to rule out CHIP).6 Considering most genetic testing is performed on leukocytes from blood (derived from mesoderm), the subsequent tissue tested is usually skin fibroblasts (mesoderm) and/or buccal cells (ectoderm). In the current case, the TP53 variant was detected at VAF of 31.1% in blood, which is right at the threshold and therefore may have been reported as a heterozygous variant or as a mosaic variant, depending on laboratory-specific criteria.
Testing of additional tissues from the patient showed the presence of this variant in all three germ layers at different VAFs: 44.6% in saliva (ectoderm), 18.3% in fibroblasts (mesoderm), and only 9.0% in healthy colon tissue (endoderm). This finding rules out CHIP and is suggestive that the variant arose at an early enough time point during development to be present in all three germ layers, albeit not significantly affecting endoderm-derived colon tissue. Ideally, obtaining tissues from each germ layer provides a more complete understanding of the distribution of the variant; however, this type of testing is limited by the tissue that may be easily sampled. When a male partner is suspected of mosaicism, testing may be carried out on their sperm cells. Testing of female ovarian tissue, however, is invasive and not as straightforward. In the current case, the absence of the TP53 variant in 10 embryos implies that our patient’s gonadal tissue (mesoderm) is likely unaffected or harbors the variant in a small proportion of cells. It is therefore not associated with a significant reproductive risk in this individual and is the most likely explanation for 10 unaffected embryos.
Why is germline mosaicism clinically important to identify?
Identification of mosaicism is important for relaying disease and reproductive risks to patients, highlighting how these may differ from patients with classic LFS or CHIP. A de novo TP53 variant that is present in all cells of an individual will have a 50% risk of inheritance. A variant resulting from CHIP is somatic, with essentially a zero-risk of inheritance. For a mosaic variant however, counselling for risk of inheritance is challenging. A variant may be present in somatic tissues alone with complete absence from gonadal tissue, which implies a low to zero risk of inheritance, similar to this case. Conversely, a variant may arise after differentiation of the primordial germ cells (embryonic precursors of sperm and eggs). In this scenario, the variant would be absent from all other tissues and only present in gonadal tissue. Standard molecular testing in blood would thus be misleadingly negative. However, the individual would have a risk of passing on the variant to their offspring, which is dependent on how many germ cells were affected.
In conclusion, interpretation of a variant identified at an allele fraction <30% warrants several considerations, including mosaicism and CHIP.6 Analysis from another tissue type can distinguish these two scenarios. However, counselling for true risk for inheritance is challenging. In light of this uncertainty, current practice guidelines recommend the same clinical management for mosaic TP53 individuals and their families as for non-mosaic heterozygous individuals.2
Nisha Kanwar, Ph.D.
Fellow, Laboratory Genetics and Genomics
Mayo Clinic
Wei Shen, Ph.D.
Consultant, Laboratory Genetics and Genomics
Mayo Clinic
Assistant Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science
