Exome sequencing in four families with neurodevelopmental disorders: genotype-phenotype correlation and identification of novel disease-causing variants in VPS13B and RELN.

Neurodevelopmental disorders (NDDs) are a clinically and genetically heterogeneous group of early-onset pediatric disorders that affect the structure and/or function of the central or peripheral nervous system. Achieving a precise molecular diagnosis for NDDs may be challenging due to the diverse genetic underpinnings and clinical variability. In the current study, we investigated the underlying genetic cause(s) of NDDs in four unrelated Pakistani families. Using exome sequencing (ES) as a diagnostic approach, we identified disease-causing variants in established NDD-associated genes in all families, including one hitherto unreported variant in RELN and three recurrent variants in VPS13B, DEGS1, and SPG11. Overall, our study highlights the potential of ES as a tool for clinical diagnosis.

Untargeted metabolomics-based identification of bioactive compounds from Mangifera indica L. seed extracts in drug discovery through molecular docking and assessment of their anticancer potential.

BACKGROUND: Mangifera indica L. (mango), a medicinal plant rich in biologically active compounds, has potential to be used in disease-preventing and health-promoting products. The present investigation reveals and uncovers bioactive metabolites with remarkable therapeutic efficiency from mango (family: Anacardiaceae) seeds. RESULTS: Biological activity was determined by antimicrobial, antioxidant and anticancer assays, and metabolite profiling was performed on gas chromatography coupled to quadrupole time-of-flight mass spectrometry (GC-QTOF-MS) and liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) platforms. Validation of active metabolites was carried out by in silico molecular docking (Molinspiration Cheminformatics Server and PASS). Extracted and identified metabolites were screened; 54 compounds associated with various groups were selected for the in silico interaction study. CONCLUSIONS: Molecular docking revealed lead molecules with a potential binding energy score, efficacy and stable modulation with a selected protein domain. Investigation, directed by in vitro and in silico analysis, confirms mango seeds as an excellent source of potential metabolites as a therapeutic agent. © 2024 Society of Chemical Industry.

Evaluation of image quality and radiation dose in computed tomography urography following tube voltage optimisation.

INTRODUCTION: Computed tomography urography (CTU) comprehensively evaluates the urinary tract. However, the procedure is associated with a high radiation dose due to multiple scan series and therefore requires optimisation. The study performed CTU protocol optimisation based on a reduction in tube voltage (kV) using quality assurance (QA) phantom and clinical images and evaluated image quality and radiation dose. METHODS: The study was prospectively conducted on patients referred for CTU. The patients were grouped into A and B and were scanned with the standard protocol, a protocol used for the routine CTU at the CT centre before optimisation, and optimised protocol, a protocol with reduced kV respectively. The protocols were first tried on a quality assurance (QA) phantom before being applied to patients, and image quality was assessed based on signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR). In addition, the clinical images were assessed based on the visibility of the anatomical criteria for CT images by five observers with >5 years of experience. The data were analysed using both visual grading characteristic (VGC) curves and statistical package for social sciences (SPSS) version 22.0. RESULTS: The dose was significantly lower in the optimised protocol with a 10 % reduction in both volume computed tomography dose index and (CTDIvol) and dose length product (DLP) for the phantom images, and a 26 % reduction in CTDIvol and 28 % in DLP for the clinical images. However, there was no significant difference in image quality noted between the standard and optimised protocols based on the quantitative and qualitative image quality evaluation using both the QA phantom and clinical images. CONCLUSION: The findings revealed a significant dose reduction in the optimised protocol. Further, image quality in standard and optimised protocols did not differ significantly based on quantitative and qualitative methods. IMPLICATION FOR PRACTICE: kV optimisation in contrast-enhanced procedures provides dose reduction and should be encouraged in the medical imaging departments.

Two-dimensional simulations of Rayleigh-Taylor instability in elastic-plastic media.

Two-dimensional numerical simulations for the Rayleigh-Taylor instability in an elastic-plastic medium are presented. Recent predictions of the theory regarding the asymmetric growth of peaks and valleys during the linear phase of the instability evolution are confirmed. Extension to the nonlinear regime reveals singular features, such as the long delay in achieving the nonlinear saturation and an intermediate phase with growth rate larger than the classical one.

Formation of spikes and bubbles in the linear phase of Rayleigh-Taylor instability in elastic-plastic media.

The generation of spikes and bubbles, a typical characteristic of the nonlinear regime in the Rayleigh-Taylor instability, is found to occur as well during the linear regime in an elastic-plastic solid medium caused, however, by a very different mechanism. This singular feature originates in the differential loads at different locations of the interface, which makes that the transition from the elastic to the plastic regime takes place at different times, thus producing an asymmetric growth of peaks and valleys that rapidly evolves in exponentially growing spikes, while bubbles can also grow exponentially at a lower rate.

Regulatory de novo mutations underlying intellectual disability.

The genetic aetiology of a major fraction of patients with intellectual disability (ID) remains unknown. De novo mutations (DNMs) in protein-coding genes explain up to 40% of cases, but the potential role of regulatory DNMs is still poorly understood. We sequenced 63 whole genomes from 21 ID probands and their unaffected parents. In addition, we analysed 30 previously sequenced genomes from exome-negative ID probands. We found that regulatory DNMs were selectively enriched in fetal brain-specific enhancers as compared with adult brain enhancers. DNM-containing enhancers were associated with genes that show preferential expression in the prefrontal cortex. Furthermore, we identified recurrently mutated enhancer clusters that regulate genes involved in nervous system development (CSMD1, OLFM1, and POU3F3). Most of the DNMs from ID probands showed allele-specific enhancer activity when tested using luciferase assay. Using CRISPR-mediated mutation and editing of epigenomic marks, we show that DNMs at regulatory elements affect the expression of putative target genes. Our results, therefore, provide new evidence to indicate that DNMs in fetal brain-specific enhancers play an essential role in the aetiology of ID.

Cylindrical convergence effects on the Rayleigh-Taylor instability in elastic and viscous media.

Convergence effects on the perturbation growth of an imploding surface separating two nonideal material media (elastic and viscous media) are analyzed in the case of a cylindrical implosion in both the Rayleigh-Taylor stable and unstable configurations. In the stable configuration, the perturbation damping effect due to angular momentum conservation becomes destroyed for sufficiently high values of the elastic modulus or of the viscosity of the media. For the unstable configuration, Rayleigh-Taylor instability can be suppressed by the elasticity or mitigated by the viscosity, but without practically affecting the perturbation growth due to the geometrical convergence. However, the convergence effects manifest themselves in a manner somewhat different from the classical Bell-Plesset effect by making the process more sensitive to the media compressibility than in the case involving ideal media.

Cost-effectiveness of Denosumab for the Treatment of Postmenopausal Osteoporosis in Malaysia.

From the perspective of Malaysian health care providers, denosumab was cost-effective in the treatment of postmenopausal osteoporosis, with an optimal outcome starting at age 60 years. Our results provide important insights into the value for money of anti-osteoporotic agents that can serve as a reference for other countries with comparable epidemiological data. INTRODUCTION: The study aimed to compare the cost-effectiveness of denosumab with alendronate and no treatment in the management of postmenopausal osteoporosis among the Malaysian population. METHODS: A well-validated Markov model was used to estimate the cost-effectiveness of denosumab in a hypothetical cohort of postmenopausal osteoporotic women between 50 and 80 years old who had no history of fractures. A 10-year time horizon from the perspective of Malaysian health care providers was used in this analysis. The model parameters, including transition probabilities and costs, were based on Malaysian sources. Treatment efficacy data were obtained from a network meta-analysis. The study outcomes were presented as incremental cost per quality-adjusted life-year (QALY) gained. Sensitivity analyses were performed to ensure the robustness of the results. A cost-effectiveness threshold was set at MYR 21,438 (USD 5175) per QALY. RESULTS: Denosumab was found to be a cost-effective option for postmenopausal osteoporotic women aged 60 and older. The incremental cost-effectiveness ratios (ICERs) for denosumab versus alendronate ranged from MYR 16,955 (USD 4093) per QALY at age 60 to MYR 4380 (USD 1057) per QALY at age 80. The cost-effectiveness of denosumab improved monotonically with increasing age. Denosumab was 72.8-92.7% likely to be cost-effective at the cost-effectiveness threshold. Sensitivity analyses demonstrated that the results were robust across all parameter variations, with the annual cost of denosumab being the most sensitive. CONCLUSIONS: From the perspective of the Malaysian health care provider, denosumab appears to be a cost-effective treatment choice for postmenopausal osteoporotic women over 60 years of age.

Imaging characteristics of infantile fibrosarcoma.

AIM: To highlight the imaging findings in a case series of histologically confirmed infantile fibrosarcoma (IF) and identify any features specific to this entity. MATERIALS AND METHODS: Retrospective identification was undertaken of patients with histologically confirmed IF from the electronic patient databases of two institutions between 1 January 2010 and 1 May 2021. Available pre-treatment imaging, histopathological reports, and clinical records were reviewed. RESULTS: Eighteen patients with IF met the inclusion criteria. There were 10 male and eight female patients with a mean age at presentation of 3 weeks. All patients had the t (12; 15) chromosomal translocation. Eleven (61%) tumours were located in the extremities, three were in the craniofacial region, two were intrathoracic, one abdominal and one paraspinal. A single patient had extensive metastases. The tumours were generally isointense to skeletal muscle on T1-weighted sequences and hyperintense on T2 with heterogeneous enhancement and high cellularity seen as diffusion restriction. Fifteen of the 18 lesions were evaluated on ultrasound and appeared as heterogeneous, hypervascular solid or mixed solid/cystic masses, mimicking benign vascular lesions in two cases. CONCLUSION: The present two-centre, retrospective study of the largest case series described thus far demonstrates that IF is always highly cellular on magnetic resonance imaging but has no other specific imaging features. It should be considered in the differential diagnosis of any enlarging soft-tissue, solid mass arising in the limbs or neck at birth or in infancy.

A Phase Ib/II Randomized Study of RO4929097, a Gamma-Secretase or Notch Inhibitor with or without Vismodegib, a Hedgehog Inhibitor, in Advanced Sarcoma.

PURPOSE: Because the Hedgehog and Notch pathways are often overexpressed in mesenchymal malignancies, we evaluated the efficacy of concurrent inhibition of Notch and Hedgehog signaling using the gamma-secretase inhibitor (GSI) RO4929097 and the smoothened antagonist vismodegib in unresectable or metastatic sarcoma. PATIENTS AND METHODS: In this investigator-initiated trial, phase Ib used standard 3+3 dose escalation in which patients first received vismodegib once daily for 21 days, followed by the combination of RO4929097 concurrently with vismodegib in 21-day cycles. In phase II, patients were randomized to RO4929097 alone or in combination with vismodegib. RESULTS: Nine patients were treated in phase Ib with no dose-limiting toxicities. RO4929097 at 15 mg daily in combination with 150 mg daily of vismodegib was declared the recommended phase II dose. Most adverse events were grade ≤ 2. In phase II (closed early due to discontinuation of RO4929097 evaluation), 34 patients were randomized to RO4929097 alone and 33 to RO4929097 plus vismodegib. RO4929097 did not interfere with the steady-state concentration of vismodegib, while vismodegib reduced the plasma concentration of RO492909. No patients had an objective response. Neither progression-free nor overall survival differed significantly between treatment arms. Paired tumor biopsies from a subset of patients demonstrated inhibition of cleaved Notch. CONCLUSIONS: The combination of RO4929097 plus vismodegib was generally well tolerated. Although accrual to this study was not completed, vismodegib did not meaningfully enhance the clinical efficacy of RO4929097 in an unplanned analysis. GSIs and GSIs plus vismodegib can inhibit intratumoral Notch and downstream phosphorylated Akt signaling.

A recurrent rare intronic variant in CAPN3 alters mRNA splicing and causes autosomal recessive limb-girdle muscular dystrophy-1 in three Pakistani pedigrees.

Autosomal recessive limb-girdle muscular dystrophy-1 (LGMDR1) is an autosomal recessive disorder characterized by progressive weakness of the proximal limb and girdle muscles. Biallelic mutations in CAPN3 are reported frequently to cause LGMDR1. Here, we describe 11 individuals from three unrelated consanguineous families that present with typical features of LGMDR1 that include proximal muscle wasting, weakness of the upper and lower limbs, and elevated serum creatine kinase. Whole-exome sequencing identified a rare homozygous CAPN3 variant near the exon 2 splice donor site that segregates with disease in all three families. mRNA splicing studies showed partial retention of intronic sequence and subsequent introduction of a premature stop codon (NM_000070.3: c.379 + 3A>G; p.Asp128Glyfs*15). Furthermore, we observe reduced CAPN3 expression in primary dermal fibroblasts derived from an affected individual, suggesting instability and/or nonsense-mediated decay of mutation-bearing mRNA. Genome-wide homozygosity mapping and single-nucleotide polymorphism analysis identified a shared haplotype and supports a possible founder effect for the CAPN3 variant. Together, our data extend the mutational spectrum of LGMDR1 and have implications for improved diagnostics for individuals of Pakistani origin.

Elastic-plastic Rayleigh-Taylor instability at a cylindrical interface.

The boundaries of stability are determined for the Rayleigh-Taylor instability at a cylindrical interface between an ideal fluid in the interior and a heavier elastic-plastic solid in the outer region. The stability maps are given in terms of the maximum dimensionless initial amplitude ξ_{th}^{*} that can be tolerated for the interface to remain stable, for any particular value of the dimensionless radius B of the surface, and for the different spatial modes m of the perturbations. In general, for the smallest dimensionless radius and larger modes m, the interface remains stable for larger values of ξ_{th}^{*}. In particular, for m>1 and B→0, it turns out ξ_{th}^{*}→1, and a cylindrical geometry equivalent to Drucker's criterion is found, which indeed ends up being independent of the interface geometry.

Growth Inhibition and Induction of Innate Immune Signaling of Chondrosarcomas with Epigenetic Inhibitors.

Chondrosarcomas are inherently resistant to chemotherapy and radiotherapy, pointing to an unmet need for new treatment options. Immune checkpoint inhibitors, which have shown remarkable promise in multiple solid cancer types, have limited efficacy in chondrosarcomas. Mutations in IDH1/2 genes, which result in progressive increases in DNA and histone methylation, are observed in 50% of conventional chondrosarcomas, suggesting that epigenetic dysregulation represents a potential barrier for tumor progression and target for therapeutic intervention. Here, we demonstrated that combined treatment of FDA-approved inhibitors of DNA methyltransferases (DNMTs) 5-aza-2'-deoxycytidine (5-aza), and histone deacetylases (HDACs) suberanilohydroxamic acid (SAHA) impaired the proliferation of chondrosarcoma cell lines in vitro and in xenograft studies. Transcriptomic analysis reveals that chondrosarcoma cells treated with 5-aza and SAHA markedly elevated the expression of IFN-stimulated genes including PD-L1, indicating that these epigenetic drugs induced a potent innate immune response. We demonstrated that 5-aza and SAHA resulted in both genomic and epigenomic instability, as shown by elevated DNA damage response and derepression of retrotransposons, respectively, which in turn activated pattern recognition receptors (PRRs) and the downstream IFN signaling pathways. Importantly, the cytotoxic effects of 5-aza and SAHA can be rescued by depletion of PRRs such as cGAS and MAVS, and potentiated by depletion of the RNA-editing enzyme ADAR1. Together, our results demonstrate preclinical activity of combined DNMT and HDAC inhibition against chondrosarcomas and suggest that targeted epigenetic therapies could represent a new therapeutic approach in the treatment of chondrosarcomas, and this is being tested in an ongoing clinical trial (NCT04340843).

Haploinsufficiency of the Sin3/HDAC corepressor complex member SIN3B causes a syndromic intellectual disability/autism spectrum disorder.

Proteins involved in transcriptional regulation harbor a demonstrated enrichment of mutations in neurodevelopmental disorders. The Sin3 (Swi-independent 3)/histone deacetylase (HDAC) complex plays a central role in histone deacetylation and transcriptional repression. Among the two vertebrate paralogs encoding the Sin3 complex, SIN3A variants cause syndromic intellectual disability, but the clinical consequences of SIN3B haploinsufficiency in humans are uncharacterized. Here, we describe a syndrome hallmarked by intellectual disability, developmental delay, and dysmorphic facial features with variably penetrant autism spectrum disorder, congenital malformations, corpus callosum defects, and impaired growth caused by disruptive SIN3B variants. Using chromosomal microarray or exome sequencing, and through international data sharing efforts, we identified nine individuals with heterozygous SIN3B deletion or single-nucleotide variants. Five individuals harbor heterozygous deletions encompassing SIN3B that reside within a ∼230 kb minimal region of overlap on 19p13.11, two individuals have a rare nonsynonymous substitution, and two individuals have a single-nucleotide deletion that results in a frameshift and predicted premature termination codon. To test the relevance of SIN3B impairment to measurable aspects of the human phenotype, we disrupted the orthologous zebrafish locus by genome editing and transient suppression. The mutant and morphant larvae display altered craniofacial patterning, commissural axon defects, and reduced body length supportive of an essential role for Sin3 function in growth and patterning of anterior structures. To investigate further the molecular consequences of SIN3B variants, we quantified genome-wide enhancer and promoter activity states by using H3K27ac ChIP-seq. We show that, similar to SIN3A mutations, SIN3B disruption causes hyperacetylation of a subset of enhancers and promoters in peripheral blood mononuclear cells. Together, these data demonstrate that SIN3B haploinsufficiency leads to a hitherto unknown intellectual disability/autism syndrome, uncover a crucial role of SIN3B in the central nervous system, and define the epigenetic landscape associated with Sin3 complex impairment.

Rayleigh-Taylor instability in elastic-plastic solid slabs bounded by a rigid wall.

The linear evolution of the incompressible Rayleigh-Taylor instability for the interface between an elastic-plastic slab medium and a lighter semi-infinite ideal fluid beneath the slab is developed for the case in which slab is attached to a rigid wall at the top surface. The theory yields the maps for the stability in the space determined by the initial perturbation amplitude and wavelength, as well as for the transition boundary from the elastic to the plastic regimes for arbitrary thicknesses of the slab and density contrasts between the media. In particular, an approximate but very accurate scaling law is found for the minimum initial perturbation amplitude required for instability and for the corresponding perturbation wavelength at which it occurs. These results allows for an interpretation of the recent experiments by Maimouni et al. [Phys. Rev. Lett. 116, 154502 (2016)PRLTAO0031-900710.1103/PhysRevLett.116.154502].

Candidate variants in TUB are associated with familial tremor.

Essential tremor (ET) is the most common adult-onset movement disorder. In the present study, we performed whole exome sequencing of a large ET-affected family (10 affected and 6 un-affected family members) and identified a TUB p.V431I variant (rs75594955) segregating in a manner consistent with autosomal-dominant inheritance. Subsequent targeted re-sequencing of TUB in 820 unrelated individuals with sporadic ET and 630 controls revealed significant enrichment of rare nonsynonymous TUB variants (e.g. rs75594955: p.V431I, rs1241709665: p.Ile20Phe, rs55648406: p.Arg49Gln) in the ET cohort (SKAT-O test p-value = 6.20e-08). TUB encodes a transcription factor predominantly expressed in neuronal cells and has been previously implicated in obesity. ChIP-seq analyses of the TUB transcription factor across different regions of the mouse brain revealed that TUB regulates the pathways responsible for neurotransmitter production as well thyroid hormone signaling. Together, these results support the association of rare variants in TUB with ET.

Studies of equation of state properties of high-energy-density matter generated by intense ion beams at the facility for antiprotons and ion research.

The work presented in this paper shows with the help of two-dimensional hydrodynamic simulations that intense heavy-ion beams are a very efficient tool to induce high energy density (HED) states in solid matter. These simulations have been carried out using a computer code BIG2 that is based on a Godunov-type numerical algorithm. This code includes ion beam energy deposition using the cold stopping model, which is a valid approximation for the temperature range accessed in these simulations. Different phases of matter achieved due to the beam heating are treated using a semiempirical equation-of-state (EOS) model. To take care of the solid material properties, the Prandl-Reuss model is used. The high specific power deposited by the projectile particles in the target leads to phase transitions on a timescale of the order of tens of nanosecond, which means that the sample material achieves thermodynamic equilibrium during the heating process. In these calculations we use Pb as the sample material that is irradiated by an intense uranium beam. The beam parameters including particle energy, focal spot size, bunch length, and bunch intensity are considered to be the same as the design parameters of the ion beam to be generated by the SIS100 heavy-ion synchrotron at the Facility for Antiprotons and Ion Research (FAIR), at Darmstadt. The purpose of this work is to propose experiments to measure the EOS properties of HED matter including studies of the processes of phase transitions at the FAIR facility. Our simulations have shown that depending on the specific energy deposition, solid lead will undergo phase transitions leading to an expanded hot liquid state, two-phase liquid-gas state, or the critical parameter regime. In a similar manner, other materials can be studied in such experiments, which will be a very useful addition to the knowledge in this important field of research.

Detection of hemophilia by fluorescence spectroscopy: A photodiagnosis approach.

Hemophilia (HP), the deficiency of clotting factors such as VIII (FVIII) and IX, is an inherited blood disorder which is due to the lack of clotting protein. Conventional techniques for detecting hemophilia are based on clotting factor tests such as hemostasis assays to determine various types of hemophilia and its severity. In the current study, we propose a new approach involving a spectral technique to discriminate normal controls from hemophilia patients with an accuracy above 80 %. This accuracy is calculated on the normalized relative intensity based on the light measurement of blood components by analyzing a certain set of fluorescent bio molecules. Our results indicated that Red Blood Cells (RBCs) show lower porphyrin content and enzyme deficiencies in hemophilia patients than in normal controls. Therefore, the spectral features in hemophilia patients are different from those of normal controls. Thus, the proposed technique is a good alternative for the detection of hemophilia patients when compared with conventional detection techniques.

Recessive variants in ZNF142 cause a complex neurodevelopmental disorder with intellectual disability, speech impairment, seizures, and dystonia.

PURPOSE: The purpose of this study was to expand the genetic architecture of neurodevelopmental disorders, and to characterize the clinical features of a novel cohort of affected individuals with variants in ZNF142, a C2H2 domain-containing transcription factor. METHODS: Four independent research centers used exome sequencing to elucidate the genetic basis of neurodevelopmental phenotypes in four unrelated families. Following bioinformatic filtering, query of control data sets, and secondary variant confirmation, we aggregated findings using an online data sharing platform. We performed in-depth clinical phenotyping in all affected individuals. RESULTS: We identified seven affected females in four pedigrees with likely pathogenic variants in ZNF142 that segregate with recessive disease. Affected cases in three families harbor either nonsense or frameshifting likely pathogenic variants predicted to undergo nonsense mediated decay. One additional trio bears ultrarare missense variants in conserved regions of ZNF142 that are predicted to be damaging to protein function. We performed clinical comparisons across our cohort and noted consistent presence of intellectual disability and speech impairment, with variable manifestation of seizures, tremor, and dystonia. CONCLUSION: Our aggregate data support a role for ZNF142 in nervous system development and add to the emergent list of zinc finger proteins that contribute to neurocognitive disorders.

Mutations in NCAPG2 Cause a Severe Neurodevelopmental Syndrome that Expands the Phenotypic Spectrum of Condensinopathies.

The use of whole-exome and whole-genome sequencing has been a catalyst for a genotype-first approach to diagnostics. Under this paradigm, we have implemented systematic sequencing of neonates and young children with a suspected genetic disorder. Here, we report on two families with recessive mutations in NCAPG2 and overlapping clinical phenotypes that include severe neurodevelopmental defects, failure to thrive, ocular abnormalities, and defects in urogenital and limb morphogenesis. NCAPG2 encodes a member of the condensin II complex, necessary for the condensation of chromosomes prior to cell division. Consistent with a causal role for NCAPG2, we found abnormal chromosome condensation, augmented anaphase chromatin-bridge formation, and micronuclei in daughter cells of proband skin fibroblasts. To test the functional relevance of the discovered variants, we generated an ncapg2 zebrafish model. Morphants displayed clinically relevant phenotypes, such as renal anomalies, microcephaly, and concomitant increases in apoptosis and altered mitotic progression. These could be rescued by wild-type but not mutant human NCAPG2 mRNA and were recapitulated in CRISPR-Cas9 F0 mutants. Finally, we noted that the individual with a complex urogenital defect also harbored a heterozygous NPHP1 deletion, a common contributor to nephronophthisis. To test whether sensitization at the NPHP1 locus might contribute to a more severe renal phenotype, we co-suppressed nphp1 and ncapg2, which resulted in significantly more dysplastic renal tubules in zebrafish larvae. Together, our data suggest that impaired function of NCAPG2 results in a severe condensinopathy, and they highlight the potential utility of examining candidate pathogenic lesions beyond the primary disease locus.