Kristen Rasmussen

Clinical genetics provider real-time workflow study

Purpose: Our work is the first documentation, in real time, of workflow in a general genetics department including data on patient care, research, and other activities for both clinical geneticists and genetic counselors.Methods: All physician geneticists and genetic counselors in the medical genetics department used an electronic tool to record their activities in 15 minute increments during clinic hours, evenings, and weekends over a 10-week period.Results: The average work week was 54.1 hours for physicians and 43.5 hours for genetic counselors. During clinic hours physicians spent about one-fourth of their time on direct patient care, one-fourth on other patient-related activities, one-fourth on research unrelated to individual patient care, and the remaining fourth on all other activities. However, after hours and on weekends they spent most of their time on research. Genetic counselors spent half of their time on patient-related activities, one-fourth on direct patient care, and the remainder on all other activities. The total professional time averaged 7 hours per new patient and 3.5 hours per follow-up with nearly 60% of this time devoted to patient-related activities.Conclusions: The labor intensive nature of clinical genetics, the large amount of time devoted to patient-related activities, and continuing limitations on billing by genetic counselors all contribute to the financial challenges faced by genetics departments.

An analysis of PAX1 in the development of vertebral malformations.

Due to the sporadic occurrence of congenital vertebral malformations, traditional linkage approaches to identify genes associated with human vertebral development are not possible. We therefore identified PAX1 as a candidate gene in vertebral malformations and congenital scoliosis due to its mutation in the undulated mouse. We performed DNA sequence analysis of the PAX1 gene in a series of 48 patients with congenital vertebral malformations, collectively spanning the entire vertebral column length. DNA sequence coding variants were identified in the heterozygous state in exon 4 in two male patients with thoracic vertebral malformations. One patient had T9 hypoplasia, T12 hemivertebrae and absent T10 pedicle, incomplete fusion of T7 posterior elements, ventricular septal defect, and polydactyly. This patient had a CCC (Pro)→CTC (Leu) change at amino acid 410. This variant was not observed in 180 chromosomes tested in the National Institute of Environmental Health Sciences (NIEHS) single nucleotide polymorphism (SNP) database and occurred at a frequency of 0.3% in a diversity panel of 1066 human samples. The second patient had a T11 wedge vertebra and a missense mutation at amino acid 413 corresponding to CCA (Pro)→CTA (Leu). This particular variant has been reported to occur in one of 164 chromosomes in the NIEHS SNP database and was found to occur with a similar frequency of 0.8% in a diversity panel of 1066 human samples. Although each patients mother was clinically asymptomatic and heterozygous for the respective variant allele, the possibility that these sequence variants have clinical significance is not excluded.

A Missense T(Brachyury) Mutation Contributes to Vertebral Malformations

No major susceptibility genes for sporadically occurring congenital vertebral malformations (CVM) in humans have been identified to date. Body patterning genes whose mutants cause axial skeletal anomalies in mice are candidates for human CVM susceptibility. T (also known as Brachyury) and TBX6 are critical genes needed to establish mesodermal identity. We hypothesized that mutations in T and/or TBX6 contribute to the pathogenesis of human CVMs. The complete T and TBX6 coding regions, splice junctions, and proximal 500 bp of the promoters were sequenced in 50 phenotyped patients with CVM. Three unrelated patients with sacral agenesis, Klippel‐Feil syndrome, and multiple cervical and thoracic vertebral malformations were heterozygous for a c.1013C>T substitution, resulting in a predicted Ala338Val missense alteration in exon 8. A clinically unaffected parent of each patient also harbored the substitution, but the variant did not occur in an ethnically diverse, 443‐person reference population. The c.1013C>T variant is significantly associated with CVM (p < 0.001). Alanine 338 shows moderate conservation across species, and valine at this position has not been reported in any species. A fourth patient harbored a c.908–8C>T variant in intron 7. This previously unreported variant was tested in 347 normal control subjects, and 11 heterozygotes and 2 T/T individuals were found. No TBX6 variants were identified. We infer that the c.1013C>T substitution is pathogenic and represents the first report of an association between a missense mutation in the T gene and the occurrence of sporadic CVMs in humans. It is uncertain whether the splice junction variant increases CVM risk. TBX6 mutations do not seem to be associated with CVM. We hypothesize that epistatic interactions between T and other developmental genes and the environment modulate the phenotypic consequences of T variants.

DLL3 as a candidate gene for vertebral malformations

Investigations have not identified a major locus for congenital vertebral malformations. Based on observations in mice, we hypothesized that mutations in DLL3, a member of the notch‐signaling pathway, might contribute to human vertebral malformations. We sequenced the DLL3 gene in 50 patients with congenital vertebral malformations. A Caucasian male patient with VACTERL manifestations including a T5–T6 block vertebrae was heterozygous for a “G” to “A” missense mutation changing glycine to arginine at codon 269. This residue is conserved in mammals, including chimpanzee, mouse, dog, and rat. Additional testing in the patient did not show evidence of chromosome abnormalities. The patients asymptomatic mother was also heterozygous for the missense mutation. Since this mutation was not observed in a control population and leads to an amino acid change, it may be clinically significant. The mutation was not found in a control population of 87 anonymous individuals. Several established mechanisms could explain the mutation in both the patient and his asymptomatic mother (susceptibility allele requiring additional environmental factors, somatic mosaicism, multigenic inheritance). Documenting the absence of the mutation in a larger control population or the presence of the mutation in additional affected patients, or documenting a functional difference in DLL3 would provide further evidence supporting its causal role.

Genetics of Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) is a genetic disorder characterized by elevated low-density lipoprotein (LDL) cholesterol and premature cardiovascular disease, with a prevalence of approximately 1 in 200–500 for heterozygotes in North America and Europe. Monogenic FH is largely attributed to mutations in the LDLR, APOB, and PCSK9 genes. Differential diagnosis is critical to distinguish FH from conditions with phenotypically similar presentations to ensure appropriate therapeutic management and genetic counseling. Accurate diagnosis requires careful phenotyping based on clinical and biochemical presentation, validated by genetic testing. Recent investigations to discover additional genetic loci associated with extreme hypercholesterolemia using known FH families and population studies have met with limited success. Here, we provide a brief overview of the genetic determinants, differential diagnosis, genetic testing, and counseling of FH genetics.

Evaluation of SLC35A3 as a candidate gene for human vertebral malformations.

Nader Ghebranious, James K. Burmester, Ingrid Glurich, Elizabeth McPherson, Lynn Ivacic, Jennifer Kislow, Kristen Rasmussen, Vikram Kumar, Cathleen L. Raggio, Robert D. Blank, F. Stig Jacobsen, Thomas Faciszewski, James Womack, and Philip F. Giampietro* Molecular Diagnostics Genotyping Laboratory, Marshfield Clinic, Marshfield, Wisconsin Center for Human Genetics, Marshfield Clinic Research Foundation, Marshfield, Wisconsin Office of Research Facilitation, Marshfield Clinic Research Foundation, Marshfield, Wisconsin Department of Medical Genetic Services, Marshfield Clinic, Marshfield, Wisconsin Department of Pediatrics, Marshfield Clinic, Marshfield, Wisconsin Department of Pediatric Orthopedics, Hospital for Special Surgery, New York, New York University of Wisconsin Medical School, Madison, Wisconsin Geriatrics Research, Education, and Clinical Center, William S. Middleton Veterans Administration Medical Center, Madison, Wisconsin Department of Orthopedic Spine Surgery, Marshfield Clinic, Marshfield, Wisconsin Veterinary Pathobiology, Texas A&M University, College Station, Texas

Lack of evidence of WNT3A as a candidate gene for congenital vertebral malformations.

BackgroundPrior investigations have not identified a major locus for vertebral malformations, providing evidence that there is genetic heterogeneity for this condition. WNT3A has recently been identified as a negative regulator of Notch signaling and somitogenesis. Mice with mutations in Wnt3a develop caudal vertebral malformations. Because congenital vertebral malformations represent a sporadic occurrence, linkage approaches to identify genes associated with human vertebral development are not feasible. We hypothesized that WNT3A mutations might account for a subset of congenital vertebral malformations.MethodsA pilot study was performed using a cohort of patients with congenital vertebral malformations spanning the entire vertebral column was characterized. DNA sequence analysis of the WNT3A gene in these 50 patients with congenital vertebral malformations was performed.ResultsA female patient of African ancestry with congenital scoliosis and a T12-L1 hemivertebrae was found to be heterozygous for a missense variant resulting in the substitution of alanine by threonine at codon 134 in highly conserved exon 3 of the WNT3A gene. This variant was found at a very low prevalence (0.35%) in a control population of 443 anonymized subjects and 1.1% in an African population.ConclusionThese data suggest that WNT3A does not contribute towards the development of congenital vertebral malformations. Factors such as phenotypic and genetic heterogeneity may underlie our inability to detect mutations in WNT3A in our patient sample.

Extreme Values of Maternal Serum Analytes in Second Trimester Screening: Looking Beyond Trisomy and NTD’s

Second trimester maternal serum screening can identify high risk pregnancies and fetuses at risk for birth defects (in addition to those in the standard interpretation). The purpose of this study was to quantify such risks to improve counseling. We compared outcomes of 692 pregnancies that had abnormal levels of at least one analyte with a cohort of 713 pregnancies with normal analytes. Increased risks include: demise with high AFP and low uE3; intrauterine growth restriction with high AFP, high and low hCG, and low uE3; placental abnormalities with high AFP; fetal stress with high AFP and high hCG. Birth defects are increased with high AFP, high hCG, and low hCG. When two or more analytes are abnormal, 46% have a poor outcome. Abnormal levels of maternal serum analytes provide information in addition to the risks for neural tube defects, Down syndrome, and trisomy 18. This information is important for counseling and pregnancy management.

Bile duct anomalies in a male child with Noonan syndrome: A case for ras and notch pathway synergism

We describe a boy with a PTPN11 mutation whose clinical features overlap both Noonan and Alagille syndrome. His clinical presentation argues for Ras and Notch pathway synergism. Delivery was by cesarean at 37/2 weeks gestation. Birth weight was 3.0 kg. At 5 weeks of age he developed cholestatic jaundice and direct hyperbilirubinemia. A liver biopsy demonstrated a paucity of intrahepatic bile ducts and inflammation (reviewed at Marshfield Clinic and Children’s Hospital of Philadelphia) (Fig. 1A). TORCH panel was negative for congenital infections. Alpha-1 antitrypsin and serum amino acid studies were within normal limits. Serum bile acids were elevated. Liver function studies and bilirubin returned to normal following treatmentwithActigall during infancy.Anechocardiogram performed because of the presence of a cardiac murmur showed mild pulmonary artery branch stenosis that resolved on follow-up echocardiogram. Speech and motor milestones were delayed, necessitating speech, physical, and occupational therapy. Several ophthalmologic evaluations were performed which failed to demonstrate embryotoxon. There was absence of butterfly vertebrae on spine radiograph. He underwent a bilateral orchiopexy at age 4 for undescended testes. In addition to the above problems, the patient also had proportionate short stature, failure to thrive, learning problems, and expressive language delay. A clinical diagnosis of Alagille syndrome was suspected. Sequence of the JAG1 gene demonstrated a heterozygous polymorphism of no known clinical significance, Y1176; TAC/TAT. Endocrine evaluation at 6/4 years of age was significant for a prealbumin of 11 mg/dl (normal 20–46) and bone age which was two standard deviations below the mean for age. He had one hospitalization for idiopathic thrombocytopenic purpura at 7/2 years of age. Physical examination at 8/2 years of age was significant for height, weight, and head circumference <3rd centile. He had proportionate short stature, a triangular-shaped face, pointed chin, hypertelorism, deeply set eyes, highly arched palate, and lentigenes distributed on his face and throughout the trunk (Fig. 1B). He has required special education instruction during his school career and has emotional problems and conduct disturbance. A diagnosis of Noonan syndrome was suspected by the attending endocrinologist due to the presence of short stature, cognitive delays, cryptorchidism, and facial dysmorphism. DNA analysis showed he had a P491S mutation in the PTPN11 gene, namely a heterozygous single base change of C!T in exon 13 consistent with a diagnosis of Noonan syndrome. This mutation has previously been documented in Noonan syndrome and LEOPARD syndrome [Zenker et al., 2004; Tartaglia et al., 2006]. This patient also fulfills the clinical criterion for LEOPARD syndrome due to the presence of lentigenes, pulmonary stenosis, growth retardation, and genital abnormalities. Since LEOPARD and Noonan syndromes are part of a phenotypic continuum and may have similar genetic etiologies,we chose todisclose a

Whole exome sequencing identifies a POLRID mutation segregating in a father and two daughters with findings of Klippel-Feil and Treacher Collins syndromes.

We report on a father and his two daughters diagnosed with Klippel–Feil syndrome (KFS) but with craniofacial differences (zygomatic and mandibular hypoplasia and cleft palate) and external ear abnormalities suggestive of Treacher Collins syndrome (TCS). The diagnosis of KFS was favored, given that the neck anomalies were the predominant manifestations, and that the diagnosis predated later recognition of the association between spinal segmentation abnormalities and TCS. Genetic heterogeneity and the rarity of large families with KFS have limited the ability to identify mutations by traditional methods. Whole exome sequencing identified a nonsynonymous mutation in POLR1D (subunit of RNA polymerase I and II): exon2:c.T332C:p.L111P. Mutations in POLR1D are present in about 5% of individuals diagnosed with TCS. We propose that this mutation is causal in this family, suggesting a pathogenetic link between KFS and TCS.