PURPOSE:Autosomal dominant polycystic kidney disease (ADPKD) affects 1:1000, causing 5% to 10% of kidney failure. The primary disease gene, PKD1, has 6 pseudogenes with 97% to 99% homology, a >12-kb transcript, high GC content, and polypyrimidine tracts. Although long-range polymerase chain reaction with Sanger sequencing has been the "gold-standard," next-generation sequencing (NGS) is increasingly used. METHODS:We performed exome sequencing (ES) on 203 ADPKD patients in 171 families from the Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease cohort, with prior "gold-standard" results: n = 157 PKD1, n = 27 PKD2, n = 19 with no pathogenic variant detected. Clinical geneticists, blinded to genotype, reviewed ES data. We assessed effects of pipeline modifications, exome capture reagents, pseudogene alignment, and deeper ES or genome sequencing (GS) for unsolved cases. RESULTS:Optimized ES identified 95.5% of defined PKD1 pathogenic variants, all PKD2 variants, and at least 9 of 19 "unsolved" cases. Standard pipelines on research-grade ES missed at least 22 PKD1 variants due to Genome Analysis Toolkit HardFiltering or alternative locus annotation. Higher-depth ES achieved 100% PKD1 variant detection. GS identified a balanced translocation t(1;16)(q31.1;p13.3). CONCLUSION:NGS matches "gold-standard" sensitivity and considers additional disease genes. ES plus GS solve 96% of well-phenotyped ADPKD. We outline practical considerations for NGS on PKD1.

Trisomy 8 mosaicism (T8M) syndrome is a rare aneuploidy condition affecting 1/25,000-50,000 live births. Affected individuals have highly variable phenotypes from very mild dysmorphism to severe structural anomalies caused by chromosomal mosaicism and possibly undetected molecular aberrations. The utilization of chromosome microarray analysis (CMA) and exome sequencing (ES) in clinical laboratories enable the identification of genomic copy number imbalances and pathogenic gene variants. We presented one patient with a double aneuploid mosaic pattern of Monosomy X and Trisomy 8 for a compound phenotype of Turner syndrome (TS) and T8M syndrome, the second patient with T8M and a mosaic pathogenic variant in the PTEN gene detected by ES, and the third patient with typical phenotypic constellation of malformations with no other genetic aberrations detected by CMA and ES. Classification of mosaic findings was provided using a recommended six-attribute scheme. Review of the literature summarized cases of T8M with concomitant molecular defects of a deletion at 22q11.2 and pathogenic variants in the SALL1, RECQL4, NF1, CASK, and PAH genes. These observations indicated that integrated cytogenetic and genomic analyses should be offered to patients with phenotypic abnormalities outside the spectrum of the T8M syndrome for comprehensive laboratory diagnosis and clinical management.

Rapid genome sequencing (rGS) has been used increasingly in clinical care in recent years due to the high diagnostic yield and shorter turnaround time. Since 2019 the DNA Diagnostic laboratory at Yale School of Medicine has been offering rGS analysis for critically ill patients admitted to the pediatric and neonatal intensive care units (ICU). The ICU team and consulting providers make the decision to test using rGS based on the likelihood of a genetic etiology and the need for an urgent diagnosis to guide clinical management.

PurposeThe specialty of Laboratory Genetics and Genomics (LGG) was created in 2017 in an effort to reflect the increasing convergence in technologies and approaches between clinical molecular genetics and clinical cytogenetics. However, there has not yet been any formal evaluation of the merging of these disciplines and the challenges faced by Program Directors (PDs) tasked with ensuring the successful training of laboratory geneticists under the new model.MethodsAn electronic multi-question Qualtrics survey was created and was sent to the PD for each of the ACGME-accredited LGG fellowship programs at the time. The data was collected and the responses were aggregated for each question.ResultsAll of the responding PDs had started training at least one LGG fellow. PDs noted challenges with funding, staff shortages, molecular/cytogenetics content integration, limited total training time, increased remote work, increased sendout testing, and a lack of prior cytogenetics knowledge among incoming fellows.ConclusionThis survey attempted to assess the challenges that LGG PDs have been facing in offering and integrating clinical molecular genetics and clinical cytogenetics fellowship training. Common challenges between programs were noted, and a set of six concluding comments are provided to facilitate future discussion.

Trisomy 8 mosaicism syndrome (T8MS), also known as Warkany syndrome 2, is a rare aneuploidy condition affecting 1/25,000–50,000 live births with male to female ratio of 5:1. Affected individuals have a highly variable phenotype varying from very mild dysmorphism to severe structural anomalies. Despite clinical variability, common features include high birth weight, accelerated somatic development, developmental delay and intellectual disability, prominent forehead, strabismus, upturned nose with broad nasal bridge, low-set dysmorphic ears, inverted lower lip, slender trunk, hypoplastic patellae, vertebral malformations, clinodactyly, and deep skin furrows on the palms and soles.

BACKGROUND:Rare variants in melanocortin 4 receptor gene (MC4R) result in a severe form of early-onset obesity; however, it is unclear how these variants may affect abdominal fat distribution, intrahepatic fat accumulation, and related metabolic sequelae. METHODS:Eight hundred seventy-seven youth (6-21 years) with overweight/obesity, recruited from the Yale Pediatric Obesity Clinic in New Haven, CT, underwent genetic analysis to screen for functionally damaging, rare variants (MAF < 0.01) in MC4R. Participants were assigned to a Pathogenic Variant or No Pathogenic Variant group and completed a 10-timepoint 180-min oral glucose tolerance test (OGTT) and abdominal MRI. RESULTS:Compared to the No Pathogenic Variant group, the Pathogenic Variant group demonstrated significantly greater glucose concentrations (AUCtot: 24.7 ± 1.22 g/dL × 180 min vs. 21.9 ± 1.41 g/dL × 180 min; p = 0.001), insulin levels (AUCtot: 57.4 ± 11.5 mU/mL × 180 min vs. 35.5 ± 8.90 mU/mL × 180 min; p = 0.002), and lower insulin sensitivity (WBISI: 1.01 ± 0.137 vs. 1.85 ± 0.036; p = 0.0008) during the OGTT. The Pathogenic Variant group also presented with greater visceral adipose tissue (VAT) (85.1 cm2 ± 10.3 vs. 56.1 cm2 ± 1.64; p = 0.003) and intrahepatic fat content (HFF%) (19.4% ± 4.94 vs. 8.21% ± 0.495; p = 0.012) than the No Pathogenic Variant group despite the two groups having similar BMI z-scores (p = 0.255), subcutaneous adipose tissue (SAT) (p = 0.643), and total body fat (p = 0.225). CONCLUSIONS:Pathogenic variants in MC4R are associated with increased VAT, HFF%, and insulin resistance, independent from the degree of obesity in youth.

Hereditary connective tissue disorders have overlapping phenotypes, particularly in regard to musculoskeletal features. This contributes to the challenge of phenotype-based clinical diagnoses. However, some hereditary connective tissue disorders have distinct cardiovascular manifestations that require early intervention and specific management. Molecular testing has increased the ability to categorize and diagnose distinct hereditary connective tissue disorders. A 42-yr-old female with a clinical diagnosis of Larsen syndrome from birth presented for genetic testing based on her recent diagnosis of premenopausal breast cancer. She had a past medical history of multiple carotid dissections. As she never had confirmatory molecular genetic testing for Larsen syndrome, whole-exome sequencing was utilized to assess both hereditary cancer predisposition syndromes and connective tissue disorders. A homozygous pathogenic variant in theFKBP14gene was identified associated withFKBP14kyphoscoliotic Ehlers–Danlos syndrome. We recommend that patients with a clinical diagnosis of Larsen syndrome undergo broad-based molecular sequencing for multiple hereditary connective tissue disorders. Molecular diagnosis is particularly crucial for all individuals who have a history of significant vascular events in the setting of a clinical diagnosis only. Early diagnosis of a hereditary connective tissue disorder with vascular features allows for screening and subsequent prevention of cardiovascular events.

X-linked hypophosphatemia (XLH), the most common form of hereditary rickets, is due to inactivation of PHEX, resulting in increased circulating fibroblast growth factor 23. Consequent renal phosphate loss leads to hypophosphatemia, rickets, and progressive bow deformity. Inheritance is X-linked dominant, such that heterozygous females are affected, as well as hemizygous males. A 10-month-old girl was referred for potential treatment for presumed XLH. Amniocentesis, performed following prenatal identification of duodenal atresia, polyhydramnios, and intrauterine growth restriction, revealed a de novo X-chromosomal deletion encompassing 10 genes, including PHEX. Postnatal genetic testing confirmed presence of the deletion in the baby. She demonstrated no phenotypic, biochemical, or radiographic features of XLH. Neither parent had features of XLH, nor carried the deletion. Given the discordance between genotype and phenotype, evaluation for skewed X-inactivation was pursued. Methylation analysis via the androgen receptor locus was inconclusive, thus RNA sequencing was pursued. Analysis of 12 high-quality single nucleotide polymorphisms (SNPs) that are expressed in mRNA revealed skewed X-inactivation. Heterozygous disruption of PHEX typically confers a diagnosis of XLH. Skewed X-inactivation, whereby one X chromosome is preferentially silenced, appears to have protected this patient from the expected expression of an X-linked dominant disorder.

Background Whole-exome sequencing (WES) is an effective tool for diagnosis in patients who remain undiagnosed despite a comprehensive clinical work-up. While WES is being used increasingly in pediatrics and oncology, it remains underutilized in non-oncological adult medicine, including in patients with liver disease, in part based on the faulty premise that adults are unlikely to harbor rare genetic variants with large effect size. Here, we aim to assess the burden of rare genetic variants underlying liver disease in adults at two major tertiary referral academic medical centers. Methods WES analysis paired with comprehensive clinical evaluation was performed in fifty-two adult patients with liver disease of unknown etiology evaluated at two US tertiary academic health care centers.Findings Exome analysis uncovered a definitive or presumed diagnosis in 33% of patients (17/52) providing insight into their disease pathogenesis, with most of these patients (12/17) not having a known family history of liver disease. Our data shows that over two-thirds of undiagnosed liver disease patients attaining a genetic diagnosis were being evaluated for cholestasis or hepatic steatosis of unknown etiology.Interpretation This study reveals an underappreciated incidence and spectrum of genetic diseases presenting in adulthood and underscores the clinical value of incorporating exome sequencing in the evaluation and management of adults with liver disease of unknown etiology.Funding S.V. is supported by the NIH/NIDDK (K08 DK113109 and R01 DK131033-01A1) and the Doris Duke Charitable Foundation Grant #2019081. This work was supported in part by NIH-funded Yale Liver Center, P30 DK34989.Copyright & COPY; 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Multiple mitochondrial enzymes employ lipoic acid as a coenzyme. Pathogenic variants in LIAS, encoding lipoic acid synthase (LIAS), are associated with autosomal recessive LIAS-related disorder (OMIM# 614462). This disorder is characterized by infantile-onset hypotonia, profound psychomotor delay, epileptic encephalopathy, nonketotic hyperglycinemia, and lactic acidosis. We present the case of a 20-year-old female who experienced developmental deficits at the age of 6 months and began to have seizures at 3 years of age. Exome sequencing revealed compound heterozygous novel variants in LIAS, designated c.277delC (p.Leu93Ter) and c.542A > T (p.Asp181Val). The p.Leu93Ter variant is predicted to cause loss of function due to the severe truncation of the encoded protein. To examine the p.Asp181Val variant, functional analysis was performed using Baker's yeast (Saccharomyces cerevisiae) lacking LIP5, the homologue of human LIAS. Wild-type LIAS promoted oxidative growth of the lip5∆ yeast strain. In contrast, lip5∆ yeast expressing p.Asp181Val exhibited poor growth, similar to known pathogenic variants, p.Asp215Glu and p.Met310Thr. Our work has expanded the phenotypic and genotypic spectrum of LIAS-related disorder and established the use of the yeast model as a system for functional study of novel missense variants in LIAS.

Supplementary Figure Legends and Tables 1-2 from Cytogenetic Instability in Ovarian Epithelial Cells from Women at Risk of Ovarian Cancer

In the last decade, the utility of whole-exome sequencing in uncovering genetic aetiologies of a variety of liver diseases has been demonstrated. These new diagnoses have guided the management, treatment, and prognostication of previously undiagnosed patients, largely thanks to improved insight into the underlying pathogenesis of their conditions. Despite its clear benefits, the uptake of genetic testing by hepatologists has been limited, in part due to limited prior genetic training and/or opportunities for continuing education. Herein, we show that Hepatology Genome Rounds, an interdisciplinary forum highlighting hepatology cases of clinical interest and educational value, are an important venue for integrating genotypic and phenotypic information to enable accurate diagnosis and appropriate management, dissemination of genomic knowledge within the field of hepatology, and ongoing education to providers and trainees in genomic medicine. We describe our single-centre experience and discuss practical considerations for clinicians interested in launching such a series. We foresee that this format will be adopted at other institutions and by additional specialties, with the aim of further incorporating genomic information into clinical medicine.

Supplementary Figure 1 from Cytogenetic Instability in Ovarian Epithelial Cells from Women at Risk of Ovarian Cancer