September 2021 – Biochemical Genetics

A 13-month-old girl presents with recurrent vomiting for three months. Her labs are significant for elevated AST, ALT, and alkaline phosphatase and prolonged INR (not specified).

Ammonia = 200 (≤80 mcmol/L) 

Lactate = 22 (normal <19mg/dL)

Amino acid panel and urine organic acid analysis showed the following:

Table 1
Table 2

Which urea cycle disorder is the most likely diagnosis?

  • N-acetylglutamate synthetase deficiency (NAGS)
  • Ornithine Transcarbamylase deficiency (OTC)
  • Carbamoyl Phosphate Synthethase deficiency (CPSI)
  • Arginosuccinate Synthase 1 Deficiency (ASS1)

The correct answer is ...

The correct answer is: Ornithine Transcarbamylase deficiency (OTC).

Ammonia levels above 200 umol/L suggests an enzyme early in the urea cycle. 

Quantitative analysis of amino acids (AA) performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) showed elevated glutamine and alanine, low arginine and citrulline.

Urine organic acids analysis showed high levels of orotic acid excretion (332 mmol/mol creatinine; reference values: <4) and uracil. There were no other unusual organic acids. 


OTC diagnosis was confirmed. Arginine supplementation and hydration were advised. 

Ornithine transcarbamylase (OTC) deficiency is a rare, X-linked genetic disorder characterized by complete or partial lack of the enzyme ornithine transcarbamylase (OTC) in which hemizygous males are almost always symptomatic and about 20% of female carriers, may present some neurocognitive deficit.

OTC is the most frequent cause of UCD, accounting for nearly 50% of all cases. OTC deficiency is the most common inherited disease of ureagenesis, with an estimated prevalence of 1 in 62,000 to 1 in 77,000


  1. Rush, Eric T et al. “Late-onset ornithine transcarbamylase deficiency: An under recognized cause of metabolic encephalopathy.” SAGE open medical case reports vol. 2 2050313X14546348. 31 Jul. 2014, doi:10.1177/2050313X14546348
  2. Savy, Nadia et al. “Acute pediatric hyperammonemia: current diagnosis and management strategies.” Hepatic medicine : evidence and research vol. 10 105-115. 12 Sep. 2018, doi:10.2147/HMER.S140711

Paola Ramos, Ph.D.

Fellow, Clinical Chemistry
Mayo Clinic

Kimiyo Raymond, M.D. 

Consultant, Laboratory Genetics and Genomics
Mayo Clinic
Assistant Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science

September 2021 – Clinical Microbiology Case 1

A 64-year-old man involved in a minor MVA was found to have a new parietal brain lesion and 5 mm pulmonary nodule on CT. He reported two months of minor upper respiratory symptoms and a two-week history of headaches with associated cognitive difficulties, weakness, and fine motor deficits. MRI of the brain showed a 2.3 cm cystic “ring enhancing” lesion (Fig. 1). Intraoperative evaluation of the brain biopsy showed fibrinous material with acute inflammation. Organism morphology on GMS (Fig. 2), modified acid-fast stain (Fig. 3), and culture medium (Fig. 4) are shown. 

Figure 1: MRI of the brain showing lesion
Figure 2: GMS
Figure 3: Modified acid-fast stain
Figure 4: Culture medium

Which of the following is true regarding this organism?

  • Calcium phosphate binds this organism together to form “sulfur granules.”
  • Colonies turn yellow after exposure to light (photochromogen).
  • It is acquired through contact with contaminated soil.
  • It is a “beaded” filamentous gram-negative bacillus.

The correct answer is ...

The correct answer is: It is acquired through contact with contaminated soil.

This is a case of Nocardiosis with pulmonary and CNS involvement. The ring enhancing lesion on MRI corresponds to the dense fibrous capsule surrounding the abscess cavity. GMS reveals clusters of filamentous organisms that display a characteristic “beaded” morphology on a modified acid-fast stain. Colonies are peach to chalky-white and grow in 3 to 5 days on Sabouraud medium or blood agar. MALDI-TOF mass spectrometry performed on positive broth culture identified Nocardia transvalensis. Due to variable resistance patterns, cultures and sensitivity testing should be performed to guide antibiotic therapy. While CNS treatment guidelines are initial drainage followed by antibiotic therapy for 12 months, recurrence after discontinuing treatment is common. Despite aggressive treatment, CNS Nocardiosis mortality rates remain high: 20% in immunocompetent patients and 55% in immunocompromised patients.

There are over 100 species of Nocardia, with at least 15 species known to infect humans, most often occurring in an immunocompromised host. They are found in soil and rotting vegetation and inhalation of contaminated dust is the most common route of exposure. On Gram stain, Nocardia spp. are “beaded” filamentous, and branching, gram-positive organisms and are partially acid-fast, differentiating them from Actinomyces spp., which are not acid-fast and characteristically produce “sulfur granules” in tissue. Colonies of Nocardia grown in culture do not require exposure to light to display the characteristic soft peach color, as opposed to photochromogenic species of mycobacteria, such as M. kansasii.


  1. Goldman, L., Schafer, A. I., & Cecil, R. L. (2020). Chapter 314: Nocardiosis. In Goldman-Cecil medicine (26th ed., pp. 2032–2034). essay, Elsevier. 
  2. Sun, H., Goolam Mahomed, M., & Patel, J. (2021). Brain metastasis or nocardiosis? A case report of central nervous system Nocardiosis with a review of the literature. Journal of community hospital internal medicine perspectives11(2), 258–262.

Holly Berg, D.O., MLS(CM)

Resident, Anatomic and Clinical Pathology
Mayo Clinic

Nancy Wengenack, Ph.D.

Consultant, Clinical Microbiology
Mayo Clinic
Assistant Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science

September 2021 – Clinical Microbiology Case 2

A 55-year-old man was admitted with symptoms of septic shock and started on broad spectrum antibiotics, which included piperacillin-tazobactam and vancomycin. A computerized tomography scan showed pneumoperitoneum, metastatic pancreatic adenocarcinoma, and a hepatic abscess that had eroded through the capsule. The abscess was treated by drain placement as the patient was deemed not a surgical candidate.

Blood cultures yielded the organism shown in the image below. The organism grew as β-hemolytic colonies on sheep blood agar. It did not grow on Thiosulfate-citrate-bile salts-sucrose (TCBS) agar. The organism tested oxidase- and indole-positive, and fermented glucose.

Photo Courtesy: Dr. Madiha Fida

Which of the following is the most likely identity of the recovered organism?

  • Listeria species
  • Enterobacteriaceae species 
  • Vibrio species
  • Aeromonas species

The correct answer is ...

The correct answer is: Aeromonas species.

Unlike Vibrio species, Aeromonas species do not grow on TCBS agar, making this agar useful in distinguishing Aeromonas from Vibrio. Enterobacteriaceae species ferments glucose and are indole positive but oxidase negative, which distinguishes it from Aeromonas. Listeria species are gram positive.


  1. Aeromonas infections. (n.d.). Retrieved from
  2. Aeromonas wound infections associated with outdoor activities -- California. (n.d.). Retrieved from
  3. 2Fernández-Bravo, A., & Figueras, M. J. (2020). An update on the genus Aeromonas: Taxonomy, epidemiology, and pathogenicity. Microorganisms, 8(1), 129. doi:10.3390/microorganisms8010129
  4. Freeman, J., & Roberts, S. (n.d.). Approach to Gram stain and culture results in the microbiology laboratory. Retrieved from
  5. 1Janda, J. M., & Abbott, S. L. (2010). The genus Aeromonas: Taxonomy, pathogenicity, and infection. Clinical Microbiology Reviews, 23(1), 35-73. doi:10.1128/cmr.00039-09
  6. Morris, J. G., Jr. (n.d.). Minor Vibrio and Vibrio-like species associated with human disease. Retrieved from

Nadarra Stokes, M.D.

Resident, Anatomic and Clinical Pathology
Mayo Clinic

Audrey Schuetz, M.D.

Consultant, Clinical Microbiology
Mayo Clinic
Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science

September 2021 – Hematopathology

A 14-year-old girl with recurrent tonsillar infections had tonsillectomy. Bilateral palatine tonsils and right lingual tonsil were excised and submitted for histologic examination. Hematoxylin and eosin and immunohistochemical stains are shown (Fig.1 and 2). 

Figure 1: IRF4
Figure 2: IRF4

If you can perform only one additional ancillary test to confirm the diagnosis, which one would you choose?

  • FISH for IRF4 rearrangement
  • In situ hybridization for EBV
  • FISH for MYC rearrangement
  • Molecular study for MYD88 mutation

The correct answer is ...

The correct answer is: FISH for IRF4 rearrangement.

The clinical scenario, neoplastic cells morphology and immunohistochemical stains (neoplastic cells express CD20, CD10, BCL6 and MUM1), suggest that the patient has large B-cell lymphoma with IRF4 rearrangement. This diagnosis needs to be distinguished from other large B-cell neoplasms because the patients have a favorable outcome after treatment. 

Incorrect answer: Molecular studies for MYD88 mutation is important when lymphoplasmacytic lymphoma is in the differential. Cells morphology and immunohistochemical profile in this cases make lymphoplasmacytic lymphoma an unlikely diagnosis.

Incorrect answer: FISH for IRF4 rearrangement should be performed first, and if the result is negative, FISH for MYC, BCL2, and BCL6 rearrangement should be considered for differential diagnosis of diffuse large B-cell lymphoma and high-grade B-cell lymphoma with MYC/BCL and/or BCL6 rearrangement.

Incorrect answer: Epstein-Barr virus has not been linked to pathogenesis of large B-cell lymphoma with IRF4 rearrangement.


  1. Swerdlow S, Campo E, Harris NL, Jaffe E, Pileri S, Stein H, Thiele J, Arber D, Hasserjian R, Le Beau M. WHO classification of tumours of haematopoietic and lymphoid tissues (Revised 4th edition). IARC: Lyon 2017.
  2. Woessmann W, Quintanilla-Martinez L. Rare mature B-cell lymphomas in children and adolescents. Hematol Oncol. 2019 Jun;37 Suppl 1:53-61. doi: 10.1002/hon.2585. PMID: 31187530.Granai M, Lazzi S. Early pattern of large B-cell lymphoma with IRF4 rearrangement. Blood. 2020 Aug 6;136(6):769. doi: 10.1182/blood.2020006406. PMID: 32761226

Krasimira Rozenova, M.D., Ph.D.

Resident, Anatomic and Clinical Pathology
Mayo Clinic

Rebecca King, M.D.

Consultant, Hematopathology
Mayo Clinic
Associate Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science

September 2021 – Molecular Pathology

Siblings, a 15-year-old girl and a 16-year-old boy, present with progressive muscle weakness. Their history is notable for delayed motor milestones and proximal muscle weakness, progressing to loss of ambulation. Spinal muscular atrophy (SMA) deletion duplication analysis reveals both siblings to have one copy of the SMN1 gene and two copies of the SMN2 gene. Results of the SMN1 full gene analysis are included in the table below.

Figure 1: SMN1 full gene analysis 

Can a definitive molecular diagnosis of SMA be made, and what other molecular tests may be needed?

  • A diagnosis of SMA can be made. The c.5C>G variant in the SMN1 or SMN2 gene is pathogenic.
  • A diagnosis of SMA can be made. The patients only have one copy of SMN1.
  • A diagnosis of SMA cannot be made. A definitive molecular diagnosis would require full gene analysis that distinguishes SMN1 exon 1 from SMN2 exon 1.
  • A diagnosis of SMA cannot be made. Further testing with Neuromuscular Genetic Panels by Next Generation Sequencing is needed.

The correct answer is ...

A molecular diagnosis of SMA cannot be made.  A definitive molecular diagnosis would require full gene analysis that distinguishes SMN1 exon 1 from SMN2 exon 1.

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease which occurs in one out of every 10,000 live births, making it the leading genetic cause of infant mortality. SMA is characterized by degeneration of the alpha motor neurons of the spinal cord leading to progressive muscle weakness, and in cases of Type I SMA, progression to respiratory failure and death before age 2 if supportive therapies are not initiated.  

However, SMA is a clinically heterogeneous disease, with Type I being the most severe of the four types. Patients with Type II SMA present with marked delay in gross motor development by 6-18 months of life, never become ambulatory, and suffer from progressive weakness, scoliosis, and restrictive lung disease. Life expectancy of SMA Type II patients ranges from 2 years of age to 40 years of age. Patients with Type III SMA have variable age of onset and clinical course. They often require wheelchair assistance by adolescence; however, their life expectancy does not significantly differ from the general population. The mildest type of SMA, Type IV, generally presents in the second or third decade of life, with mild proximal limb girdle weakness that progresses slowly.

All four types of SMA are caused by a mutation or deletion in the survival motor neuron 1 (SMN1) gene. Humans also have the survival motor neuron 2 (SMN2) gene, which differs from SMN1 gene by five nucleotides.  All of these nucleotides are in non-coding regions, except for the cysteine to thymine variation in exon 7. Ultimately, this variation is translationally silent and the amino acid sequence of SMN2 is identical to SMN1; however, the variation affects the alternative splicing such that 90% of transcripts are missing exon 7 and result in an unstable protein that is rapidly degraded. Accordingly, there is an inverse relationship between SMN2 copy number and disease severity. In general, type I SMA patients have two copies of SMN2, type II SMA patients have three copies of SMN2, and type III SMA patients have three or four copies of SMN2.

Molecular analysis of SMA patients has become increasingly important, as there are now three new therapies that have recently been FDA-approved for the treatment of SMA. The SMN1 gene exon 7 is homozygously absent in approximately 95% of affected patients. The great majority of the remainder of SMA cases is heterozygous for the exon 7 deletion and a small more subtle mutation in the other allele; compound heterozygotes. SMA cases resulting from homozygous point mutations are exceedingly rare. Therefore, the first-line molecular analysis for the diagnosis of SMA consists of detecting the homozygous loss of exon 7 of the SMN1 gene. This can be done using techniques such as MLPA, qPCR, or ddPCR, which typically detect the presence of exon 7 of the SMN1 gene, thereby quantifying the number of copies of the SMN1 gene. If the result from these techniques is inconclusive or negative, it is recommended to perform full gene analysis of the SMN1 gene. This analysis typically consists of long-range-PCR of SMN1 exons 2-8, followed by bidirectional Sanger sequence analysis. SMN1 exon 1 is also PCR-amplified and bidirectionally Sanger-sequenced; however, the primers used to amplify SMN1 exon 1 will also amplify SMN2 exon 1. Therefore, if a pathogenic variant is detected in exon 1, a definitive molecular diagnosis cannot be made. An individual with one copy of SMN1 and a variant in exon 1 of SMN2 gene does not have SMA, whereas an individual with one copy of SMN1 with a pathologic variant in exon 1 of SMN1 has SMA. 

In this case, both siblings tested positive for an exon 1 variant. Due to the limitations of the test, it cannot be determined if this variant is in SMN1 or SMN2. However, both siblings have the same variant and both have clinical histories suggestive of SMA. Therefore, a clinical diagnosis can be made. Importantly, with this diagnosis, both patients are eligible for disease modifying therapy. Of note, there are reports of efficient long-range PCR methods that are able to amplify exons 1-8 of SMN1. This method would be able to distinguish pathogenic variants in exon 1 of SMN 1 from variants in exon 1 of SMN2.


  1. Burghes, A., Beattie, C. Spinal muscular atrophy: why do low levels of survival motor neuron protein make motor neurons sick? Nat Rev Neurosci 2009; 10, 597–609.
  2. Bürglen L, Lefebvre S, Clermont O, et al. Structure and organization of the human survival motor neurone (SMN) gene. Genomics. 1996;32(3):479-482. 
  3. Clermont O, Burlet P, Benit P, et al: Molecular analysis of SMA patients without homozygous SMN1 deletions using a new strategy for identification of SMN1 subtle mutations. Hum Mutat 2004; 24:417-427.
  4. Faravelli I, Nizzardo M, Comi GP, Corti S. Spinal muscular atrophy--recent therapeutic advances for an old challenge. Nat Rev Neurol. 2015; 11(6):351-9.
  5. Feldkötter M, Schwarzer V, Wirth R, Wienker TF, Wirth B. Quantitative analyses of SMN1 and SMN2 based on real-time lightCycler PCR: fast and highly reliable carrier testing and prediction of severity of spinal muscular atrophy. Am J Hum Genet. 2002; 70(2):358-368.
  6. Kubo Y, Nishio H, Saito K: A new method for SMN1 and hybrid SMN gene analysis in spinal muscular atrophy using long-range PCR followed by sequencing. J Hum Genet 2015; 60: 233-239.
  7. Lefebvre S, Bürglen L, Reboullet S, et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell. 1995; 80(1):155-165.
  8. Mahajan R. Onasemnogene Abeparvovec for Spinal Muscular Atrophy: The Costlier Drug Ever. Int J Appl Basic Med Res. 2019; 9(3):127-128.
  9. Pearn J. Incidence, prevalence, and gene frequency studies of chronic childhood spinal muscular atrophy. J Med Genet. 1978; 15(6):409-13.
  10. Prior TW. Carrier screening for spinal muscular atrophy. Genet Med 2008;10:840-2.
  11. Prior T, Finanger E: Spinal Muscular Atrophy. Gene Reviews. Update Dec 22, 2016. 
  12. Prior TW, Krainer AR, Hua Y, et al. A positive modifier of spinal muscular atrophy in the SMN2 gene. Am J Hum Genet. 2009; 85(3):408-413.
  13. Ruhno C, McGovern VL, Avenarius MR, et al. Complete sequencing of the SMN2 gene in SMA patients detects SMN gene deletion junctions and variants in SMN2 that modify the SMA phenotype. Hum Genet. 2019; 138(3):241-256.
  14. Shorrock, H.K., Gillingwater, T.H. & Groen, E.J.N. Overview of Current Drugs and Molecules in Development for Spinal Muscular Atrophy Therapy. Drugs 2018; 78, 293–305.
  15. Sugarman EA, Nagan N, Zhu H, Akmaev VR, Zhou Z, Rohlfs EM, Flynn K, Hendrickson BC, Scholl T, Sirko-Osadsa DA, Allitto BA. Pan-ethnic carrier screening and prenatal diagnosis for spinal muscular atrophy: clinical laboratory analysis of >72,400 specimens. Eur J Hum Genet. 2012; 20(1):27-32. 
  16. Vidal-Folch N, Gavrilov D, Raymond K, Rinaldo P, Tortorelli S, Matern D, Oglesbee D. Multiplex Droplet Digital PCR Method Applicable to Newborn Screening, Carrier Status, and Assessment of Spinal Muscular Atrophy. Clin Chem.2018; 64(12):1753-1761.
  17. Wirth B: An update of the mutation spectrum of the survival motor neuron gene (SMN1) in autosomal recessive spinal muscular atrophy (SMA). Hum Mutat 2000; 15:228-237.Wu X, Wang SH, Sun J, Krainer AR, Hua Y, Prior TW. A-44G transition in SMN2 intron 6 protects patients with spinal muscular atrophy. Hum Mol Genet. 2017; 26(14):2768-2780.

Sara Cook, M.D., Ph.D.

Resident, Anatomic and Clinical Pathology
Mayo Clinic

Daniel Anderson, D.O.

Senior Associate Consultant, Neurology
Mayo Clinic

MCL Education (@mmledu)

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