Pavel N. Pichurin

Outcome of Whole Exome Sequencing for Diagnostic Odyssey Cases of an Individualized Medicine Clinic: The Mayo Clinic Experience

OBJECTIVE To describe the experience and outcome of performing whole-exome sequencing (WES) for resolution of patients on a diagnostic odyssey in the first 18 months of an individualized medicine clinic (IMC). PATIENTS AND METHODS The IMC offered WES to physicians of Mayo Clinic practice for patients with suspected genetic disease. DNA specimens of the proband and relatives were submitted to WES laboratories. We developed the Genomic Odyssey Board with multidisciplinary expertise to determine the appropriateness for IMC services, review WES reports, and make the final decision about whether the exome findings explain the disease. This study took place from September 30, 2012, to March 30, 2014. RESULTS In the first 18 consecutive months, the IMC received 82 consultation requests for patients on a diagnostic odyssey. The Genomic Odyssey Board deferred 7 cases and approved 75 cases to proceed with WES. Seventy-one patients met with an IMC genomic counselor. Fifty-one patients submitted specimens for WES testing, and the results have been received for all. There were 15 cases in which a diagnosis was made on the basis of WES findings; thus, the positive diagnostic yield of this practice was 29%. The mean cost per patient for this service was approximately

Gastrointestinal Findings in the Largest Series of Patients With Hereditary Biallelic Mismatch Repair Deficiency Syndrome: Report from the International Consortium

8000. Medicaid supported 27% of the patients, and 38% of patients received complete or partial insurance coverage. CONCLUSION The significant diagnostic yield, moderate cost, and notable health marketplace acceptance for WES compared with conventional genetic testing make the former method a rational diagnostic approach for patients on a diagnostic odyssey.

PMS2 monoallelic mutation carriers: the known unknown

Objectives:Hereditary biallelic mismatch repair deficiency (BMMRD) is caused by biallelic mutations in the mismatch repair (MMR) genes and manifests features of neurofibromatosis type 1, gastrointestinal (GI) polyposis, and GI, brain, and hematological cancers. This is the first study to characterize the GI phenotype in BMMRD using both retrospective and prospective surveillance data.Methods:The International BMMRD Consortium was created to collect information on BMMRD families referred from around the world. All patients had germline biallelic MMR mutations or lack of MMR protein staining in normal and tumor tissue. GI screening data were obtained through medical records with annual updates.Results:Thirty-five individuals from seven countries were identified with BMMRD. GI data were available on 24 of 33 individuals (73%) of screening age, totaling 53 person-years. The youngest age of colonic adenomas was 7, and small bowel adenoma was 11. Eight patients had 19 colorectal adenocarcinomas (CRC; median age 16.7 years, range 8–25), and 11 of 18 (61%) CRC were distal to the splenic flexure. Eleven patients had 15 colorectal surgeries (median 14 years, range 9–25). Four patients had five small bowel adenocarcinomas (SBC; median 18 years, range 11–33). Two CRC and two SBC were detected during surveillance within 6–11 months and 9–16 months, respectively, of last consecutive endoscopy. No patient undergoing surveillance died of a GI malignancy. Familial clustering of GI cancer was observed.Conclusions:The prevalence and penetrance of GI neoplasia in children with BMMRD is high, with rapid development of carcinoma. Colorectal and small bowel surveillance should commence at ages 3–5 and 8 years, respectively.

Pregnancy in an individual with mild Smith-Lemli-Opitz syndrome

Germ-line mutations in MLH1, MSH2, MSH6, and PMS2 have been shown to cause Lynch syndrome. The penetrance of the cancer and tumor spectrum has been repeatedly studied, and multiple professional societies have proposed clinical management guidelines for affected individuals. Several studies have demonstrated a reduced penetrance for monoallelic carriers of PMS2 mutations compared with the other mismatch repair (MMR) genes, but clinical management guidelines have largely proposed the same screening recommendations for all MMR gene carriers. The authors considered whether enough evidence existed to propose new screening guidelines specific to PMS2 mutation carriers with regard to age at onset and frequency of colonic screening. Published reports of PMS2 germ-line mutations were combined with unpublished cases from the authors’ research registries and clinical practices, and a discussion of potential modification of cancer screening guidelines was pursued. A total of 234 monoallelic PMS2 mutation carriers from 170 families were included. Approximately 8% of those with colorectal cancer (CRC) were diagnosed before age 30, and each of these tumors presented on the left side of the colon. As it is currently unknown what causes the early onset of CRC in some families with monoallelic PMS2 germline mutations, the authors recommend against reducing cancer surveillance guidelines in families found having monoallelic PMS2 mutations in spite of the reduced penetrance.Genet Med 18 1, 13–19.

The prevalence of diseases caused by lysosome-related genes in a cohort of undiagnosed patients

Smith-Lemli-Opitz syndrome (SLOS) is a recessive disorder characterized by multiple anomalies (cleft palate, postaxial polydactyly, 2/3 toe syndactyly, hypospadias, and cardiac defects), microcephaly, growth restriction, and intellectual disability. SLOS is caused by mutations in the DHCR7 gene, which encodes 7-dehydrocholesterol reductase. Decreased enzyme activity results in low levels of plasma cholesterol and elevated cholesterol precursors, including 7-dehydrocholesterol (7DHC), which is diagnostic of SLOS. The molecular mutation detection rate is as high as 96% (1). Here, we describe pregnancy in an individual with SLOS for the first time. Our patient is a 36-year-old referred to genetics during her second pregnancy for recurrence risk of her minor congenital anomalies. The patients first pregnancy was achieved with clomiphene ovulation induction and intrauterine insemination with donor sperm, and resulted in a spontaneous abortion at approximately four to five weeks gestation. Her second pregnancy was achieved spontaneously. The patient has a history of a submucosal cleft palate and “fused baby teeth.” Physical examination revealed a small mouth and high-pitched voice. The hands were small but without any digital anomalies. The feet were also small, and there was almost complete cutaneous syndactyly between the second and third toes bilaterally (Figure 1). The patient had short stature (height 147.7 cm, less than third percentile). Her weight was 73.0 kg, head circumference was 51.5 cm, BSA was 1.8 m2, and BMI was 33.5 kg/m2. She had normal intellect, with no reported history of learning disabilities, developmental delays, or behavioral abnormalities. She completed high school and college education without need for special education or tutoring. Figure 1 Patients feet. Note bilateral 2/3 toe syndactyly. SLOS was suspected given the patients congenital anomalies. 7DHC plasma levels were elevated on several occasions, including postpartum time points (Table I). The patient was not treated with any drugs that cause elevated 7DHC (e.g. Aripiprazole). Molecular testing of DHCR7 revealed the common c.964-1G>C mutation, and a novel missense variant, c.1083C>G (p.Phe361Leu), not reported in dbSNP. The phenylalanine residue is conserved across species. The patients father was unavailable for testing, but the patients mother tested positive for the c.964-1G>C mutation and negative for the c.1083C>G variant, strongly suggesting that the two DHCR7 alterations are in trans and therefore likely causative of the patients phenotype. Table 1 Results of biochemical testing performed during and after the patients pregnancy. We measured sterols in fibroblasts grown under cholesterol replete (FBS) and cholesterol deficient (LPDS) conditions (performed at NIH, Bethesda, MD) (2). When patient skin fibroblasts were cultured in cholesterol depleted medium, the mean fraction of 7DHC to total sterols was 1.8%. These values are consistent with the mildest identified patients we have identified to date. Cholesterol levels are typically elevated during pregnancy (3); however, this is not expected in SLOS. Due to the patients personal and family history of hypercholesterolemia, APOB and LDLR gene analysis were conducted. The patient tested negative for the common R3500W and R3500Q APOB mutations. LDLR gene sequencing revealed two copies of the c.1773C>T polymorphism. This polymorphism results in decreased splicing efficiency of exon 12, leading to increased total and LDL-cholesterol (4). We postulate that this polymorphism has a protective effect by counteracting the SLOS-associated cholesterol synthesis defect, explaining the patients mild phenotype. Guidelines for management of SLOS during pregnancy do not exist due to the lack of reported cases. The diagnosis of SLOS in our patient resulted in modifications of her medical care. Changes in management included serial monitoring of cholesterol and 7DHC levels with dietary intervention to increase exogenous cholesterol and decrease the accumulation of potentially toxic cholesterol precursors. The patient met with a dietician for specific dietary management recommendations. A cholesterol loading diet, including lean meats, cheese, and eggs, was recommended beginning at approximately 29 weeks gestation. Serial fetal ultrasounds were performed due to concern for intrauterine growth restriction, heart defects, or other anomalies associated with either SLOS or toxicity due to cholesterol precursors crossing the placenta. An obstetrical anesthesia consult was also ordered due to obesity and history of cleft palate. We followed this patient throughout her pregnancy. She underwent cesarean section at term due to breech presentation with delivery of a non-dysmorphic, healthy female. Short fallopian tubes were incidentally noted (6.5 cm versus 9.0-11.0 cm average). SLOS is a complex condition with a variable phenotype. Although pregnancy has not been previously reported in a patient with SLOS, mild cases may be under-ascertained. Our patient had mild physical anomalies of SLOS without cognitive abnormalities. The presence of LDLR polymorphisms in our patient may have a modulating role in cholesterol biogenesis and as such resulted in an attenuated biochemical and clinical phenotype.

Novel de novo variant in EBF3 is likely to impact DNA binding in a patient with a neurodevelopmental disorder and expanded phenotypes: patient report, in silico functional assessment, and review of published cases

Lysosomal diseases (LD) comprise a group of approximately 60 hereditary conditions caused by progressive accumulation of metabolites due to defects in lysosomal enzymes and degradation pathways, which lead to a wide range of clinical manifestations. The estimated combined incidence of LD is between 1 in 4000 to 1 in 13,000 live births, with recent data from pilot newborn screening studies showing even higher incidence. We aimed to determine the prevalence of the classical LD and other diseases caused by lysosome-related genes in our cohort of diagnostic odyssey patients. The Individualized Medicine Clinic at Mayo Clinic is increasingly utilizing whole exome sequencing (WES) to determine the genetic etiology of undiagnosed Mendelian disease. From September 2012 to April 2017, WES results from 350 patients with unexplained symptoms were reviewed. Disease-causing variants were identified in MYO6, CLN6, LRBA, KCTD7, and ARSB revealing a genetic diagnosis of a LD in 8 individuals from 5 families. Based on our findings, lysosome-related disorders may be collectively common, reaching up to 1.5% prevalence in a cohort of patients with undiagnosed diseases presenting to a genetics clinic.

Pathogenic Variant in ACTB, p.Arg183Trp, Causes Juvenile-Onset Dystonia, Hearing Loss, and Developmental Delay without Midline Malformation

Pathogenic variants in EBF3 were recently described in three back-to-back publications in association with a novel neurodevelopmental disorder characterized by intellectual disability, speech delay, ataxia, and facial dysmorphisms. In this report, we describe an additional patient carrying a de novo missense variant in EBF3 (c.487C>T, p.(Arg163Trp)) that falls within a conserved residue in the zinc knuckle motif of the DNA binding domain. Without a solved structure of the DNA binding domain, we generated a homology-based atomic model and performed molecular dynamics simulations for EBF3, which predicted decreased DNA affinity for p.(Arg163Trp) compared with wild-type protein and control variants. These data are in agreement with previous experimental studies of EBF1 showing the paralogous residue is essential for DNA binding. The conservation and experimental evidence existing for EBF1 and in silico modeling and dynamics simulations to validate comparable behavior of multiple variants in EBF3 demonstrates strong support for the pathogenicity of p.(Arg163Trp). We show that our patient presents with phenotypes consistent with previously reported patients harboring EBF3 variants and expands the phenotypic spectrum of this newly identified disorder with the additional feature of a bicornuate uterus.

Malignant Peripheral Nerve Sheath Tumor in a Patient With BAP1 Tumor Predisposition Syndrome

ACTB encodes the β-actin, and pathogenic variations in this gene have typically been associated with Baraitser-Winter cerebrofrontofacial syndrome, a congenital malformation syndrome characterized by short stature, craniofacial anomalies, and cerebral anomalies. Here, we describe the third case with the p.Arg183Trp variant in ACTB causing juvenile-onset dystonia. Our patient has severe, intractable dystonia, developmental delay, and sensorineural hearing loss, besides hyperintensities in the caudate nuclei and putamen on the brain MRI, which is a distinct but overlapping phenotype with the previously reported case of identical twins with the same alteration in ACTB.

Evidence for Brainstem Motor Nondecussation as a Neurologic Substrate for Periodic Alternating Gaze Deviation

BACKGROUND Germline pathogenic variants in BRCA1-associated protein-1 (BAP1), a nuclear ubiquitin carboxy-terminal hydrolase with evidence suggestive of independent tumor suppressor function, predispose affected families to uveal melanoma, cutaneous melanoma, renal cell carcinoma, malignant mesothelioma, and possibly a range of other tumors and malignancies as part of the BAP1 tumor predisposition syndrome, a recently recognized hereditary cancer syndrome. CASE DESCRIPTION A 50-year-old woman presented with a malignant peripheral nerve sheath tumor of the left fifth metatarsal head. Further examination revealed a right renal mass and left breast mass. Her family history was significant for astrocytoma, melanoma, cholangiocarcinoma, hepatocellular carcinoma, renal cell carcinoma, prostate cancer, non-Hodgkin lymphoma, and pancreatic adenocarcinoma. Genetic testing revealed a BAP1 mutation in the proband. CONCLUSIONS Although there have been reports of sarcomas and meningiomas in patients affected with BAP1 mutations, to our knowledge malignant peripheral nerve sheath tumors in this patient population have not been previously reported. We report a case of malignant peripheral nerve sheath tumor in a patient affected by a BAP1 mutation.

Utility of DNA, RNA, Protein, and Functional Approaches to Solve Cryptic Immunodeficiencies

A 4-month-old boy was referred for apparent inability to track and make eye contact. The parents noted that both eyes had seemed to be looking off to either side since birth. He had been born full term weighing 7 lb, 3 oz after a normal pregnancy. There was no family history of neurologic disease. He had a small skin tag on his left fifth finger. Neuro-ophthalmologic examination showed normal behavioral responses to light with an inability to follow small targets or optokinetic responses. Both pupils reacted briskly with no afferent pupillary defect. Throughout the examination, the eyes repeatedly moved from extreme right gaze to extreme left gaze with a periodicity of 10 to 20 seconds. He had no associated nystagmus and he remained conscious during these episodes. This alternating conjugate deviation persisted when the eyes were manually opened during sleep. Horizontal vestibulo-ocular reflexes were reduced. No associated nystagmus or head movements were present. Penlight and retinal examinations disclosed no abnormalities. Neurologic examination showed mild generalized hypotonia. Magnetic resonance imaging showed mild cerebellar vermian hypoplasia with thin and closely spaced superior cerebellar peduncles but no “molar tooth” sign. The cerebellar hemispheres were malrotated, with apposition of horizontal fissures in the midline and lateralization of dentate nuclei. Additional subtle signs included a borderline small pontomesencephalic junction and slightly prominent cisterna magna. Diffusion tensor imaging showed normal decussation of the transverse pontine fibers with only rudimentary decussation of superior cerebellar peduncles within the midbrain but nondecussation of the medullary pyramids (Fig 1). Two months later, the child had gained head control and now jerked his head to allow fixation in his deviated position of gaze (Video 1, available at www.aaojournal.org). These head movements were preceded by a few intermittent head jerks (Fig 1). At age 18 months, his gross motor development was delayed, and he had just begun to walk with support. Fine motor coordination was normal; he could stack several blocks in a tower and also scribble. Speech, cognition, and social development were also normal. During visual activity, he performed frequent head thrusts that preceded and were now admixed with smaller amplitudes of alternating gaze deviation (Video 2, available at www.aaojournal.org). At times, he could now fixate in primary position. A comprehensive ciliopathy gene panel was negative for an identifiable mutation for Joubert syndrome and revealed several variants of uncertain significance including in ZNF423 and TTC21B genes (both maternally inherited). With rare exceptions, periodic alternating gaze deviation is associated with Joubert syndrome. However, this infant did not display the periodic alternating skew deviation and lateral alternating skew deviation that usually accompany Joubert syndrome. Like patients with Joubert syndrome, he had periodic alternating gaze deviation with congenital ocular motor apraxia. Genetic testing did not identify mutations in genes associated with Joubert syndrome. He also lacked the pathognomonic molar tooth sign on magnetic resonance imaging, which is considered to be pathognomonic for Joubert syndrome, but showed subtle midbrainehindbrain anomalies, absent corticospinal tract decussation, and rudimentary superior cerebellar peduncle decussation, which also accompany Joubert syndrome. Given that the nondecussated corticospinal tracts subserve motor control of the limbs, we believe that disruptions of superior cerebellar peduncle decussation provided the proximate cause for the periodic alternating gaze deviation and congenital ocular motor apraxia in this infant. In contrast with classic cases of Joubert syndrome, however, the transverse pontine decussations were preserved. Like other infants with this condition, he could not track visual objects early in infancy because he could not control his eye movements at the motor level. A recent study showed that brainstem nondecussation was present in infants with congenital ocular motor apraxia and Joubert syndrome, but not in cases of “isolated” congenital ocular motor apraxia. Based on study of the human cerebellum by high angular resolution diffusion tractography, the superior, middle, and inferior cerebellar peduncles are first detectable by 17 weeks of gestational age, suggesting that the embryologic disruptions in this process occur in the first trimester of gestation. In the absence of a molar tooth sign, this case provides evidence that brainstem nondecussation may provide the neurologic substrate for periodic alternating gaze deviation. It may be that brainstem nondecussation produces a disconnection syndrome, interfering with neural feedback loops so that 1 side of the brainstem can no longer inhibit the other. Brainstem nondecussation may lead to desynchronization and reverberation within the prenuclear ocular motor circuitry. It may also provide a neural substrate for the congenital ocular motor apraxia seen in this condition. For this reason, diffusion tensor magnetic resonance imaging should be obtained to evaluate for brainstem nondecussation in children who present with periodic alternating gaze deviation.