A De Novo Deleterious PHEX Variant Without Clinical Features of X-Linked Hypophosphatemia.

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.

Advancing diagnosis and management of liver disease in adults through exome sequencing.

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.

FKBP14 kyphoscoliotic Ehlers-Danlos syndrome misdiagnosed as Larsen syndrome: a case report.

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 the FKBP14 gene was identified associated with FKBP14 kyphoscoliotic 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.

Hepatology Genome Rounds: An interdisciplinary approach to integrate genomic data into clinical practice.

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.

A retrospective cohort analysis of the Yale pediatric genomics discovery program.

The Pediatric Genomics Discovery Program (PGDP) at Yale uses next-generation sequencing (NGS) and translational research to evaluate complex patients with a wide range of phenotypes suspected to have rare genetic diseases. We conducted a retrospective cohort analysis of 356 PGDP probands evaluated between June 2015 and July 2020, querying our database for participant demographics, clinical characteristics, NGS results, and diagnostic and research findings. The three most common phenotypes among the entire studied cohort (n = 356) were immune system abnormalities (n = 105, 29%), syndromic or multisystem disease (n = 103, 29%), and cardiovascular system abnormalities (n = 62, 17%). Of 216 patients with final classifications, 77 (36%) received new diagnoses and 139 (64%) were undiagnosed; the remaining 140 patients were still actively being investigated. Monogenetic diagnoses were found in 67 (89%); the largest group had variants in known disease genes but with new contributions such as novel variants (n = 31, 40%) or expanded phenotypes (n = 14, 18%). Finally, five PGDP diagnoses (8%) were suggestive of novel gene-to-phenotype relationships. A broad range of patients can benefit from single subject studies combining NGS and functional molecular analyses. All pediatric providers should consider further genetics evaluations for patients lacking precise molecular diagnoses.

Detecting regions of homozygosity improves the diagnosis of pathogenic variants and uniparental disomy in pediatric patients.

Chromosomal microarray analysis using single nucleotide polymorphism probes can detect regions of homozygosity (ROH). This confers a potential utility in revealing autosomal recessive (AR) diseases and uniparental disomy (UPD). Results of genetic testing among pediatric patients from 2015 to 2019 were evaluated. Diagnostic findings with detected ROH from large consecutive case series in the literature were reviewed. Of 2050 pediatric patients, 65 (3%) had one or more ROH and 31 (53%) had follow-up whole exome sequencing (WES) and methylation studies. Seven homozygous variants were detected and four of them from three patients (9.6%) were within the detected ROH and classified as pathogenic or likely pathogenic variants for AR diseases. One patient (3%) had segmental UPD15q for a diagnosis of Prader-Willi syndrome. Additive diagnostic yield from ROH reporting was at least 0.2% (4/2050) of pediatric patients. These results were consistent with findings from several large case series reported in the literature. Detecting ROH had an estimated baseline predictive value of 10% for AR diseases and 3% for UPD. Consanguinity revealed by multiple ROH was a strong predictor for AR diseases. These results provide evidence for genetic counseling and recommendation of follow-up WES and methylation studies for pediatric patients reported with ROH.

D-bifunctional protein deficiency caused by splicing variants in a neonate with severe peroxisomal dysfunction and persistent hypoglycemia.

D-bifunctional protein (DBP) deficiency is a rare, autosomal recessive peroxisomal enzyme deficiency resulting in a high burden of morbidity and early mortality. Patients with DBP deficiency resemble those with a severe Zellweger phenotype, with neonatal hypotonia, seizures, craniofacial dysmorphisms, psychomotor delay, deafness, blindness, and death typically within the first 2 years of life, although patients with residual enzyme function can survive longer. The clinical severity of the disease depends on the degree of enzyme deficiency. Loss-of-function variants typically result in no residual enzyme activity; however, splice variants may result in protein with residual function. We describe a full-term newborn presenting with hypotonia, seizures, and unexplained hypoglycemia, who was later found to have rickets at follow up. Rapid whole genome sequencing identified two HSD17B4 variants in trans; one likely pathogenic variant and one variant of uncertain significance (VUS) located in the polypyrimidine tract of intron 13. To determine the functional consequence of the VUS, we analyzed RNA from the patient's father with RNA-seq which showed skipping of Exon 14, resulting in a frameshift mutation three amino acids from the new reading frame. This RNA-seq analysis was correlated with virtually absent enzyme activity, elevated very-long-chain fatty acids in fibroblasts, and a clinically severe phenotype. Both variants are reclassified as pathogenic. Due to the clinical spectrum of DBP deficiency, this provides important prognostic information, including early mortality. Furthermore, we add persistent hypoglycemia to the clinical spectrum of the disease, and advocate for the early management of fat-soluble vitamin deficiencies to reduce complications.

Detection of cytogenomic abnormalities by OncoScan microarray assay for products of conception from formalin-fixed paraffin-embedded and fresh fetal tissues.

BACKGROUND: The OncoScan microarray assay (OMA) using highly multiplexed molecular inversion probes for single nucleotide polymorphism (SNP) loci enabled the detection of cytogenomic abnormalities of chromosomal imbalances and pathogenic copy number variants (pCNV). The small size of molecular inversion probes is optimal for SNP genotyping of fragmented DNA from fixed tissues. This retrospective study evaluated the clinical utility of OMA as a uniform platform to detect cytogenomic abnormalities for pregnancy loss from fresh and fixed tissues of products of conception (POC). RESULTS: Fresh specimens of POC were routinely subjected to cell culture and then analyzed by karyotyping. POC specimens with a normal karyotype (NK) or culture failure (CF) and from formalin-fixed paraffin-embedded (FFPE) tissues were subjected to DNA extraction for OMA. The abnormality detection rate (ADR) by OMA on 94 cases of POC-NK, 38 cases of POC-CF, and 35 cases of POC-FFPE tissues were 2% (2/94), 26% (10/38), and 57% (20/35), respectively. The detected cytogenomic abnormalities of aneuploidies, triploidies and pCNV accounted for 50%, 40% and 10% in POC-CF and 85%, 10% and 5% in POC-FFPE, respectively. False negative result from cultured maternal cells and maternal cell contamination were each detected in one case. OMA on two cases with unbalanced structural chromosome abnormalities further defined genomic imbalances and breakpoints. CONCLUSION: OMA on POC-CF and POC-FFPE showed a high diagnostic yield of cytogenomic abnormalities. This approach circumvented the obstacles of CF from fresh specimens and fragmented DNA from fixed tissues and provided a reliable and effective platform for detecting cytogenomic abnormalities and monitoring true fetal result from maternal cell contamination.

Rho-GTPase Activating Protein myosin MYO9A identified as a novel candidate gene for monogenic focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a podocytopathy leading to kidney failure, whose molecular cause frequently remains unresolved. Here, we describe a rare MYO9A loss of function nonsense heterozygous mutation (p.Arg701∗) as a possible contributor to disease in a sibling pair with familial FSGS/proteinuria. MYO9A variants of uncertain significance were identified by whole exome sequencing in a cohort of 94 biopsy proven patients with FSGS. MYO9A is an unconventional myosin with a Rho-GAP domain that controls epithelial cell junction assembly, crosslinks and bundles actin and deactivates the small GTPase protein encoded by the RHOA gene. RhoA activity is associated with cytoskeleton regulation of actin stress fiber formation and actomyosin contractility. Myo9A was detected in mouse and human podocytes in vitro and in vivo. Knockin mice carrying the p.Arg701∗MYO9A (Myo9AR701X) generated by gene editing developed proteinuria, podocyte effacement and FSGS. Kidneys and podocytes from Myo9AR701X/+ mutant mice revealed Myo9A haploinsufficiency, increased RhoA activity, decreased Myo9A-actin-calmodulin interaction, impaired podocyte attachment and migration. Our results indicate that Myo9A is a novel component of the podocyte cytoskeletal apparatus that regulates RhoA activity and podocyte function. Thus, Myo9AR701X/+ knock-in mice recapitulate the proband FSGS phenotype, demonstrate that p.R701X Myo9A is an FSGS-causing mutation in mice and suggest that heterozygous loss-of-function MYO9A mutations may cause a novel form of human autosomal dominant FSGS. Hence, identification of MYO9A pathogenic variants in additional individuals with familial or sporadic FSGS is needed to ascertain the gene contribution to disease.

Exome sequencing analysis on products of conception: a cohort study to evaluate clinical utility and genetic etiology for pregnancy loss.

PURPOSE: Pregnancy loss ranging from spontaneous abortion (SAB) to stillbirth can result from monogenic causes of Mendelian inheritance. This study evaluated the clinical application of exome sequencing (ES) in identifying the genetic etiology for pregnancy loss. METHODS: A cohort of 102 specimens from products of conception (POC) with normal karyotype and absence of pathogenic copy-number variants were selected for ES. Abnormality detection rate (ADR) and variants of diagnostic value correlated with SAB and stillbirth were evaluated. RESULTS: ES detected 6 pathogenic variants, 16 likely pathogenic variants, and 17 variants of uncertain significance favor pathogenic (VUSfp) from this cohort. The ADR for pathogenic and likely pathogenic variants was 22% and reached 35% with the inclusion of VUSfp. The ADRs of SAB and stillbirth were 36% and 33%, respectively. Affected genes included those associated with multisystem abnormalities, neurodevelopmental disorders, cardiac anomalies, skeletal dysplasia, metabolic disorders, and renal diseases. CONCLUSION: These results supported the clinical utility of ES for detecting monogenic etiology of pregnancy loss. The identification of disease-associated variants provided information for follow-up genetic counseling of recurrence risk and management of subsequent pregnancies. Discovery of novel variants could provide insight for underlying molecular mechanisms causing fetal death.

A Novel Variant in the Calcium-Sensing Receptor Associated with Familial Hypocalciuric Hypercalcemia and Low-to-Normal PTH.

Familial hypocalciuric hypercalcemia (FHH) is considered a relatively benign condition characterized by mild elevations in serum calcium and relatively low urinary calcium excretion. It results from an elevated set point in serum calcium arising from variants in the calcium-sensing receptor (CaSR) gene but also AP2S1 and GNA11 genes, which encode for adaptor-related protein complex 2 and G11 proteins, respectively. The manifestations of FHH can vary and sometimes overlap with primary hyperparathyroidism making the diagnosis challenging. Case Presentations. We report a mother and daughter with a novel heterozygous variant in the CaSR gene resulting in a serine to leucine substitution at position 147 (S147L) of the CaSR. Both patients had mild hypercalcemia, relatively low urinary calcium excretion, elevated calcitriol, and low-to-normal intact PTH. The proband (daughter) presented with symptoms associated with hypercalcemia and was incidentally found to have a bony lesion suspicious for osteitis fibrosa cystica, and she was also diagnosed with sarcoidosis. Subtotal parathyroidectomy revealed normal-weight parathyroid glands comprised of 50-80% parathyroid epithelial cells, which has been documented as within the spectrum of normal. Her mother had no symptoms, and no intervention was pursued. Conclusion. We report a novel variant in the CaSR associated with FHH in two patients with similar biochemical features yet differing clinical manifestations. While the relationship of the bony findings and parathyroid histology with this variant remains unclear, these cases enrich our knowledge of CaSR physiology and provide further examples of how varied the manifestations of FHH can be.

Genes Associated with Thoracic Aortic Aneurysm and Dissection: 2019 Update and Clinical Implications.

Thoracic aortic aneurysm is a typically silent disease characterized by a lethal natural history. Since the discovery of the familial nature of thoracic aortic aneurysm and dissection (TAAD) almost 2 decades ago, our understanding of the genetics of this disorder has undergone a transformative amplification. To date, at least 37 TAAD-causing genes have been identified and an estimated 30% of the patients with familial nonsyndromic TAAD harbor a pathogenic mutation in one of these genes. In this review, we present our yearly update summarizing the genes associated with TAAD and the ensuing clinical implications for surgical intervention. Molecular genetics will continue to bolster this burgeoning catalog of culprit genes, enabling the provision of personalized aortic care.

A Retrospective Analysis of 10-Year Data Assessed the Diagnostic Accuracy and Efficacy of Cytogenomic Abnormalities in Current Prenatal and Pediatric Settings.

Background: Array comparative genomic hybridization (aCGH), karyotyping and fluorescence in situ hybridization (FISH) analyses have been used in a clinical cytogenetic laboratory. A systematic analysis on diagnostic findings of cytogenomic abnormalities in current prenatal and pediatric settings provides approaches for future improvement. Methods: A retrospective analysis was performed on abnormal findings by aCGH, karyotyping, and FISH from 3,608 prenatal cases and 4,509 pediatric cases during 2008-2017. The diagnostic accuracy was evaluated by comparing the abnormality detection rate (ADR) and the relative frequency (RF) of different types of cytogenomic abnormalities between prenatal and pediatric cases. A linear regression correlation between known prevalence and ADR of genomic disorders was used to extrapolate the prevalence of other genomic disorders. The diagnostic efficacy was estimated as percentage of detected abnormal cases by expected abnormal cases from served population. Results: The composite ADR for numerical chromosome abnormalities, structural chromosome abnormalities, recurrent genomic disorders, and sporadic pathogenic copy number variants (pCNVs) in prenatal cases were 13.03%, 1.77%, 1.69%, and 0.9%, respectively, and were 5.13%, 2.84%, 7.08%, and 2.69% in pediatric cases, respectively. The chromosomal abnormalities detected in prenatal cases (14.80%) were significantly higher than that of pediatric cases (7.97%) (p < 0.05), while the pCNVs detected in prenatal cases (2.59%) were significantly lower than that of pediatric cases (9.77%) (p < 0.05). The prevalence of recurrent genomic disorders and total pCNVs was estimated to be 1/396 and 1/291, respectively. Approximately, 29% and 35% of cytogenomic abnormalities expected from the population served were detected in current prenatal and pediatric diagnostic practice, respectively. Conclusion: For chromosomal abnormalities, effective detection of Down syndrome (DS) and Turner syndrome (TS) and under detection of sex chromosome numerical abnormalities in both prenatal and pediatric cases were noted. For pCNVs, under detection of pCNVs in prenatal cases and effective detection of DiGeorge syndrome (DGS) and variable efficacy in detecting other pCNVs in pediatric cases were noted. Extend aCGH analysis to more prenatal cases with fetal ultrasonographic anomalies, enhanced non-invasive prenatal (NIPT) testing screening for syndromic genomic disorders, and better clinical indications for pCNVs are approaches that could improve diagnostic yield of cytogenomic abnormalities.

Clinical utility of genomic analysis in adults with idiopathic liver disease.

BACKGROUND & AIMS: Adult patients suffering from liver disease of unknown cause represent an understudied and underserved population. The use of whole-exome sequencing (WES) for the assessment of a broader spectrum of non-oncological diseases, among adults, remains poorly studied. We assessed the utility of WES in the diagnosis and management of adults with unexplained liver disease despite comprehensive evaluation by a hepatologist and with no history of alcohol overuse. METHODS: We performed WES and deep phenotyping of 19 unrelated adult patients with idiopathic liver disease recruited at a tertiary academic health care center in the US. RESULTS: Analysis of the exome in 19 cases identified 4 monogenic disorders in 5 unrelated adults. Patient 1 suffered for 18 years from devastating complications of undiagnosed type 3 familial partial lipodystrophy due to a deleterious heterozygous variant in PPARG. Molecular diagnosis enabled initiation of leptin replacement therapy with subsequent normalization of liver aminotransferases, amelioration of dyslipidemia, and decreases in daily insulin requirements. Patients 2 and 3 were diagnosed with MDR3 deficiency due to recessive mutations in ABCB4. Patient 4 with a prior diagnosis of non-alcoholic steatohepatitis was found to harbor a mitochondrial disorder due to a homozygous pathogenic variant in NDUFB3; this finding enabled initiation of disease preventive measures including supplementation with antioxidants. Patient 5 is a lean patient with hepatic steatosis of unknown etiology who was found to have a damaging heterozygous variant in APOB. CONCLUSIONS: Genomic analysis yielded an actionable diagnosis in a substantial number (∼25%) of selected adult patients with chronic liver disease of unknown etiology. This study supports the use of WES in the evaluation and management of adults with idiopathic liver disease in clinical practice. LAY SUMMARY: We performed whole-exome sequencing in 19 adult patients with unexplained liver disease after an unrevealing conventional work-up performed by a hepatologist. In 5 cases, genomic analysis led to a diagnosis and informed treatment and management of the disease. Therefore, we suggest using whole-exome sequencing in the evaluation and management of adults with unexplained liver disease.

Global gene expression of histologically normal primary skin cells from BCNS subjects reveals "single-hit" effects that are influenced by rapamycin.

Studies of dominantly heritable cancers enabled insights about tumor progression. BCNS is a dominantly inherited disorder that is characterized by developmental abnormalities and postnatal neoplasms, principally BCCs. We performed an exploratory gene expression profiling of primary cell cultures derived from clinically unaffected skin biopsies of BCNS gene-carriers (PTCH1 +/-) and normal individuals. PCA and HC of untreated keratinocytes or fibroblasts failed to clearly distinguish BCNS samples from controls. These results are presumably due to the common suppression of canonical HH signaling in vitro. We then used a relaxed threshold (p-value <0.05, no FDR cut-off; FC 1.3) that identified a total of 585 and 857 genes differentially expressed in BCNS keratinocytes and fibroblasts samples, respectively. A GSEA identified pancreatic ß cell hallmark and mTOR signaling genes in BCNS keratinocytes, whereas analyses of BCNS fibroblasts identified gene signatures regulating pluripotency of stem cells, including WNT pathway. Significantly, rapamycin treatment (FDR<0.05), affected a total of 1411 and 4959 genes in BCNS keratinocytes and BCNS fibroblasts, respectively. In contrast, rapamycin treatment affected a total of 3214 and 4797 genes in normal keratinocytes and normal fibroblasts, respectively. The differential response of BCNS cells to rapamycin involved 599 and 1463 unique probe sets in keratinocytes and fibroblasts, respectively. An IPA of these genes in the presence of rapamycin pointed to hepatic fibrosis/stellate cell activation, and HIPPO signaling in BCNS keratinocytes, whereas mitochondrial dysfunction and AGRN expression were uniquely enriched in BCNS fibroblasts. The gene expression changes seen here are likely involved in the etiology of BCCs and they may represent biomarkers/targets for early intervention.

Two siblings with a novel nonsense variant provide further delineation of the spectrum of recessive KLHL7 diseases.

Mutations in Kelch-like family member 7 (KLHL7) have recently been described as a cause of a constellation of clinical findings with descriptions of both a Crisponi syndrome (CS)/cold-induced sweating syndrome type 1 (CISS1)-like, as well as a Bohring-Opitz syndrome (BOS)-like presentation. Here we report two siblings of Guatelmalan descent with a novel homozygous nonsense mutation (p.Arg326*) in KLHL7. These children have multiple dysmorphic features and developmental delay. Interestingly, their clinical traits inconsistently overlap both the CS/CISS1-like and BOS-like phenotypes, and the siblings also have subtle differences from each other, suggesting that clinicians need to be aware of the degree of variability in the presentations of these patients. Still, there is enough in common between patients with recessive KLHL7 mutations to define a novel multisystem disease that features various neurodevelopmental, musculoskeletal, dysmorphic, and other unique components. This report adds to the clinical features and disease-associated variants of the newly-recognized spectrum of KLHL7 mutations, and offers a new description, PERCHING, for the resulting syndrome.

A homozygous variant in RRM2B is associated with severe metabolic acidosis and early neonatal death.

RRM2B encodes the crucial p53-inducible ribonucleotide reductase small subunit 2 homolog (p53R2), which is required for DNA synthesis throughout the cell cycle. Mutations in this gene have been associated with a lethal mitochondrial depletion syndrome. Here we present the case of an infant with a novel homozygous p.Asn221Ser mutation in RRM2B who developed hypotonia, failure to thrive, sensorineural hearing loss, and severe metabolic lactic acidosis, ultimately progressing to death at 3 months of age. Through molecular modeling using the X-ray crystal structure of p53R2, we demonstrate that this mutation likely causes disruption of a highly conserved helix region of the protein by altering intramolecular interactions. This report expands our knowledge of potential pathogenic RRM2B mutations as well as our understanding of the molecular function of p53R2 and its role in the pathogenesis of mitochondrial DNA depletion.