Paediatric dominant and non-dominant handgrip reference curves and the association with body composition.

BACKGROUND: Lack of paediatric reference data limits the utility of handgrip strength as a measure of fitness and well-being. AIM: To develop paediatric handgrip reference curves and evaluate associations with body size and composition and race/ethnicity group. SUBJECTS AND METHODS: Handgrip, body size and composition data were obtained from National Health and Nutrition Examination Survey 2011-2014 participants aged 6-20 years. Densitometry-derived fat and appendicular lean soft tissue mass index Z-scores (FMIZ, ALSTMIZ) were generated in participants >8 years. Dominant and non-dominant handgrip reference curves were created using the LMS method. Analyses included sample weights to produce nationally representative estimates. RESULTS: Differences in handgrip strength according to hand dominance increased with age. Handgrip strength was associated with height and arm length Z-scores (R = 0.42 to 0.47) and ALSTMIZ (R = 0.54). Handgrip strength was higher in the non-Hispanic Black group and lower in the Mexican American compared to non-Hispanic White group. Group differences were attenuated when adjusted for height, arm length or ALSTMIZ. CONCLUSION: Paediatric handgrip reference curves were generated from which individual Z-scores can be calculated separately for dominant versus non-dominant hand and adjusted for body size. Association with ALSTMIZ suggests handgrip Z-score may be used as a measure of functional body composition.

Pathogenic variants in PIK3CA are associated with clinical phenotypes of kaposiform lymphangiomatosis, generalized lymphatic anomaly, and central conducting lymphatic anomaly.

Complex lymphatic anomalies are debilitating conditions characterized by aberrant development of the lymphatic vasculature (lymphangiogenesis). Diagnosis is typically made by history, examination, radiology, and histologic findings. However, there is significant overlap between conditions, making accurate diagnosis difficult. Recently, genetic analysis has been offered as an additional diagnostic modality. Here, we describe four cases of complex lymphatic anomalies, all with PIK3CA variants but with varying clinical phenotypes. Identification of PIK3CA resulted in transition to a targeted inhibitor, alpelisib. These cases highlight the genetic overlap between phenotypically diverse lymphatic anomalies.

Diagnostic stewardship for Clostridioides difficile testing in an acute care hospital: A quality improvement intervention.

Objective: To evaluate the impact of a diagnostic stewardship intervention on Clostridioides difficile healthcare-associated infections (HAI). Design: Quality improvement study. Setting: Two urban acute care hospitals. Interventions: All inpatient stool testing for C. difficile required review and approval prior to specimen processing in the laboratory. An infection preventionist reviewed all orders daily through chart review and conversations with nursing; orders meeting clinical criteria for testing were approved, orders not meeting clinical criteria were discussed with the ordering provider. The proportion of completed tests meeting clinical criteria for testing and the primary outcome of C. difficile HAI were compared before and after the intervention. Results: The frequency of completed C. difficile orders not meeting criteria was lower [146 (7.5%) of 1,958] in the intervention period (January 10, 2022-October 14, 2022) than in the sampled 3-month preintervention period [26 (21.0%) of 124; P < .001]. C. difficile HAI rates were 8.80 per 10,000 patient days prior to the intervention (March 1, 2021-January 9, 2022) and 7.69 per 10,000 patient days during the intervention period (incidence rate ratio, 0.87; 95% confidence interval, 0.73-1.05; P = .13). Conclusions: A stringent order-approval process reduced clinically nonindicated testing for C. difficile but did not significantly decrease HAIs.

Adipocytokines and Associations With Abnormal Body Composition in Rheumatoid Arthritis.

OBJECTIVE: We determined associations between adipokines and abnormal body composition in patients with rheumatoid arthritis (RA). METHODS: Combining data from three RA cohorts, whole-body dual-energy absorptiometry measures of appendicular lean mass and fat mass indices were converted to age-, sex-, and race- and ethnicity-specific Z scores. Lean mass relative to fat mass was determined based on prior methods. Independent associations between body composition profiles and circulating levels of adiponectin, leptin, and fibroblast growth factor (FGF)-21 were assessed using linear and logistic regression models adjusting for demographic characteristics and study cohort. We also determined the improvement in the area under the curve (AUC) for prediction of low lean mass when adipokines were added to predictive models that included clinical factors such as demographic characteristics, study, and body mass index (BMI). RESULTS: Among 419 participants, older age was associated with higher levels of all adipokines, whereas higher C-reactive protein level was associated with lower adiponectin levels and higher FGF-21 levels. Greater fat mass was strongly associated with lower adiponectin levels and higher leptin and FGF-21 levels. Higher levels of adiponectin, leptin, and FGF-21 were independently associated with low lean mass. The addition of adiponectin and leptin levels to regression models improved prediction of low lean mass when combined with demographic characteristics, study, and BMI (AUC 0.75 vs. 0.66). CONCLUSION: Adipokines are associated with both excess adiposity and low lean mass in patients with RA. Improvements in the prediction of body composition abnormalities suggest that laboratory screening could help identify patients with altered body composition who may be at greater risk of adverse outcomes.

Associations Between Low Serum Urate, Body Composition, and Mortality.

OBJECTIVE: Controversy remains as to whether low serum urate or uric acid (UA) levels contribute to adverse outcomes. We evaluated the relation between low serum UA levels and sarcopenia and assessed whether sarcopenia confounds associations between these low levels and mortality. METHODS: We utilized data from the National Health and Nutrition Examination Survey (1999-2006). Participants with available whole-body dual x-ray absorptiometry body composition measurements and serum UA concentrations were included. Body composition assessments included body mass index (BMI), waist circumference, maximum lifetime BMI, and age-, sex-, and race-specific appendicular lean mass index (ALMI) and fat mass index (FMI) Z scores. We also calculated Z scores for ALMI relative to FMI (ALMIFMI ). We evaluated associations between serum UA levels and body composition using logistic regression and assessed associations between serum UA levels and mortality before and after adjusting for differences in body composition using Cox proportional hazards regression. RESULTS: Among the 13,979 participants, low serum UA concentrations (<2.5 mg/dl in women, <3.5 mg/dl in men) were associated with low lean mass (ALMI and ALMIFMI Z scores), underweight BMI (<18.5 kg/m2 ), and higher rates of weight loss. The proportion of patients with low ALMI Z scores was 29% in the low serum UA group and 16% in the normal serum UA group (P = 0.001). Low serum UA levels were associated with increased mortality before we adjusted for body composition (hazard ratio 1.61 [95% confidence interval 1.14-2.28]; P = 0.008) but was attenuated and not significant after adjustment for body composition and weight loss (hazard ratio 1.30 [95% confidence interval 0.92-1.85], P = 0.13). CONCLUSION: Sarcopenia and weight loss are more common among patients with low serum UA concentrations. Differences in body composition may help to explain associations between low levels of serum UA and higher mortality.

Glycemic Control and Bone in Diabetes.

PURPOSE OF REVIEW: This review summarizes recent developments on the effects of glycemic control and diabetes on bone health. We discuss the foundational cellular mechanisms through which diabetes and impaired glucose control impact bone biology, and how these processes contribute to bone fragility in diabetes. RECENT FINDINGS: Glucose is important for osteoblast differentiation and energy consumption of mature osteoblasts. The role of insulin is less clear, but insulin receptor deletion in mouse osteoblasts reduces bone formation. Epidemiologically, type 1 (T1D) and type 2 diabetes (T2D) associate with increased fracture risk, which is greater among people with T1D. Accumulation of cortical bone micro-pores, micro-vascular complications, and AGEs likely contribute to diabetes-related bone fragility. The effects of youth-onset T2D on peak bone mass attainment and subsequent skeletal fragility are of particular concern. Further research is needed to understand the effects of hyperglycemia on skeletal health through the lifecycle, including the related factors of inflammation and microvascular damage.

Bone Mineral Density and Current Bone Health Screening Practices in Friedreich's Ataxia.

Introduction: Friedreich's Ataxia (FRDA) is a progressive neurological disorder caused by mutations in both alleles of the frataxin (FXN) gene. Impaired bone health is a complication of other disorders affecting mobility, but there is little information regarding bone health in FRDA. Methods: Dual energy X-ray absorptiometry (DXA) scan-based assessments of areal bone mineral density (aBMD) in individuals with FRDA were abstracted from four studies at the Children's Hospital of Philadelphia (CHOP). Disease outcomes, including the modified FRDA Rating Scale (mFARS), were abstracted from the FRDA Clinical Outcomes Measures Study (FACOMS), a longitudinal natural history study. A survey regarding bone health and fractures was sent to individuals in FACOMS-CHOP. Results: Adults with FRDA (n = 24) have lower mean whole body (WB) (-0.45 vs. 0.33, p = 0.008) and femoral neck (FN) (-0.71 vs. 0.004, p = 0.02) aBMD Z-scores than healthy controls (n = 24). Children with FRDA (n = 10) have a lower WB-less-head (-2.2 vs. 0.19, p < 0.0001) and FN (-1.1 vs. 0.04, p = 0.01) aBMD than a reference population (n = 30). In adults, lower FN aBMD correlated with functional disease severity, as reflected by mFARS (R = -0.56, p = 0.04). Of 137 survey respondents (median age 27 y, 50% female), 70 (51%) reported using wheelchairs as their primary ambulatory device: of these, 20 (29%) reported a history of potentially pathologic fracture and 11 (16%) had undergone DXA scans. Conclusions: Low aBMD is prevalent in FRDA, but few of even the highest risk individuals are undergoing screening. Our findings highlight potential missed opportunities for the screening and treatment of low aBMD in FRDA.

Relationship Between Serum Brain-Type Natriuretic Peptide and Biomarkers of Growth in Infants With Shunt-Dependent Single Cardiac Ventricle.

For infants with shunt-dependent or ductal-dependent single ventricle heart disease, poor growth is common and associated with morbidity and impaired neurodevelopmental outcomes. Although attention has focused on nutrition to promote weight gain, little is known about the relation between heart failure and growth factors. A prospective observational pilot study was performed to assess the relation between heart failure, assessed by brain natriuretic peptide (BNP), and growth factors (insulin-like growth factor 1 [IGF-1] and insulin-like growth factor-binding protein 3) at 3 visits: (1) before discharge from neonatal intervention with the establishment of stable pulmonary blood flow, (2) immediately before superior cavopulmonary connection, and (3) before discharge after superior cavopulmonary connection operation. The relation between BNP and growth factors was analyzed using Spearman pairwise correlations at each visit and modeled over time with a linear mixed-effects model. Correlations were considered worthy of further exploration using a p <0.10, given the exploratory nature of the study. The study included 38 infants (66% male, 68% hypoplastic left heart syndrome). Median BNP was elevated at visit 1 and decreased over time (287 pg/dl [interquartile range 147 to 794], 85 pg/dl [52 to 183], and 90 pg/dl [70 to 138]). Median IGF-1 Z score was <0 at each visit but increased over time (-0.9 [interquartile range -1.1 to 0.1], -0.7 [-1.2 to 0.1], and -0.5 [-1.2 to 0]). Inverse correlations were found between BNP and IGF-1 at visit 1 (r = -0.40, p = 0.097), BNP and IGF-1 and insulin-like growth factor-binding protein 3 at visit 2 (r = -0.33, p = 0.080 and r = -0.33, p = 0.085, respectively) and BNP and IGF-1 Z score at visit 3 (r = -0.42, p = 0.049). Significant relations were likewise found between the change in BNP and the change in IGF-1 between visits 1 and 3 (p = 0.046) and between visits 2 and 3 (p = 0.048). In conclusion, this pilot study demonstrates an inverse correlation between BNP and growth factors, suggesting that the heart failure state associated with this physiology may play a mechanistic role in impaired growth.

Treatment of cystic fibrosis related bone disease.

The advent of highly effective CFTR modulator therapies has slowed the progression of pulmonary complications in people with cystic fibrosis. There is increased interest in cystic fibrosis bone disease (CFBD) due to the increasing longevity of people with cystic fibrosis. CFBD is a complex and multifactorial disease. CFBD is a result of hypomineralized bone leading to poor strength, structure and quality leading to susceptibility to fractures. The development of CFBD spans different age groups. The management must be tailored to each group with nuance and based on available guidelines while balancing therapeutic benefits to risks of long-term use of bone-active medication. For now, the mainstay of treatment includes bisphosphonates. However, the long-term effects of bisphosphonate treatment in people with CF are not fully understood. We describe newer agents available for osteoporosis treatment. Still, the lack of data behooves trials of monoclonal antibodies treatments such as Denosumab and Romozosumab and anabolic bone therapy such as teriparatide and Abaloparatide. In this review, we also summarize screening and non-pharmacologic treatment of CFBD and describe the various options available for the pharmacotherapy of CFBD. We address the prospect of CFTR modulators on bone health while awaiting long-term trials to describe the effects of these medications on bone health.

An Update on Vitamin D Deficiency in the twenty-first century: nature and nurture.

PURPOSE OF REVIEW: Here, we review the most up-to-date understanding of the pathogenesis, prevention and treatment of vitamin D deficient rickets in children. This will include recent advances in the genetic determinants of abnormal vitamin D metabolism, with the intention of aiding clinicians with establishing the diagnosis and implementing treatment plans for children presenting with vitamin D deficiency rickets. RECENT FINDINGS: Vitamin D deficiency rickets is a frequently encountered, but entirely preventable, disorder of bone mineral metabolism. Risk factors for developing vitamin D deficiency rickets include inadequate exposure to sunlight, exclusive breast feeding without vitamin D supplementation and inadequate intake of vitamin D, calcium or phosphorus. Other factors that may influence the development of vitamin D deficiency and/or rickets include genetic alterations or medications that alter vitamin D metabolism. SUMMARY: Vitamin D levels in individuals are influenced by environmental factors, as well as genetic factors. A thorough understanding of these factors is critical for the evaluation and treatment of a child presenting with rickets. There remains a great need for additional research to determine ideal vitamin D status across diverse populations, and to better understand how vitamin D status affects overall health.

Female Sex and Obesity Are Risk Factors for Inadequate Calcium Intake in Youth With Type 1 Diabetes.

People with type 1 diabetes (T1D) are at increased risk of developing low bone mineral density and fractures. Optimization of calcium intake is a key component of pediatric bone health care. Despite the known risk factors for impaired bone health in T1D and the known benefits of calcium on bone accrual, there are limited data describing calcium intake in youth with T1D. In this cross-sectional study, calcium intake was assessed in 238 youth with T1D. One third of study participants were found to have inadequate calcium intake. Female sex, especially during adolescence, and obesity were identified as specific risk factors for inadequate calcium intake. Given the known adverse effects of T1D on bone health, efforts to promote calcium intake in youth with T1D should be considered.

Bone accrual in children and adolescents with type 1 diabetes: current knowledge and future directions.

PURPOSE OF REVIEW: Skeletal fragility is now recognized as a significant complication of type 1 diabetes (T1D). Many patients with T1D develop the disease in childhood and prior to the attainment of peak bone mass and strength. This manuscript will review recent studies investigating the effects of T1D on skeletal development. RECENT FINDINGS: Mild-to-moderate deficits in bone density, structure, and mineral accrual were reported early in the course of T1D in some but not all studies. Childhood-onset disease was associated with a more severe skeletal phenotype in some adult studies. Lower than expected bone mass for muscle size was been described. Hemoglobin A1c was negatively associated with bone density and structure in several studies, though the mechanism was not clear. SUMMARY: The use of advanced imaging techniques has shown that the adverse effects of T1D on the developing skeleton extend beyond bone density to include abnormalities in bone size, shape, microarchitecture, and strength. Despite these gains, a uniform understanding of the pathophysiology underlying skeletal fragility in this disorder remains elusive. Longitudinal studies, especially in association with interventions to reduce hyperglycemia or improve muscle strength, are needed to inform bone healthcare in T1D.

A quality improvement project to address the challenges surrounding zoledronic acid use in children.

INTRODUCTION: Zoledronic acid (ZA) is an intravenous bisphosphonate used to treat pediatric osteoporosis. Adverse events including hypocalcemia and acute phase reaction (APR) are common following first-infusion. The purpose of this report is to describe implementation of a ZA clinical practice guideline and the subsequent process changes to improve adherence to aspects of the protocol related to safety and efficacy. METHODS: Quality assurance was evaluated by chart review over a 5-year period to compare the prevalence of hypocalcemia and APR to published data. A quality improvement (QI) initiative consisting of process changes including the addition of an endocrine RN to coordinate infusions and a shift to patient/family self-scheduling of infusions was conducted. The effect of the interventions on safety (completion of pre- and post-infusion bloodwork) and efficacy (receipt of all prescribed infusions) outcomes was evaluated. RESULTS: Seventy-two patients received 244 infusions over the period. The frequency of hypocalcemia (22%) and APR (31%) was consistent with prior reports. 99% of patients received pre-infusion bloodwork, 78% received post-first-infusion bloodwork, and 47% received all prescribed infusions. QI initiatives increased the percentage of patients receiving post-first-infusion bloodwork from 67 to 79% and those receiving all infusions from 62 to 74%, but fell short of the goal of 90%. CONCLUSIONS: The implementation of a standardized protocol for ZA use in children was successful in confirming patient eligibility with pre-infusion bloodwork but failed to ensure that patients obtained post-first-infusion bloodwork and received all prescribed infusions. Further efforts to systematize the management of children on ZA are needed.

Severe multisystem organ dysfunction in an adolescent with simultaneous presentation of Addison's and Graves' disease.

OBJECTIVES: To report an unusual case of simultaneous presentation of Addison's and Graves' disease in an adolescent female previously diagnosed with type 1 diabetes (T1D) and Hashimoto's. CASE PRESENTATION: A 15-year-old female with T1D and hypothyroidism presented to the emergency department with altered mental state, fever, and left arm weakness for one day. Clinical work-up revealed coexistent new-onset adrenal insufficiency and hyperthyroidism. Her clinical course was complicated by severe, life-threating multisystem organ dysfunction including neurologic deficits, acute kidney injury, and fluid overload. Thyroidectomy was ultimately performed in the setting of persistent signs of adrenal crises and resulted in rapid clinical improvement. CONCLUSIONS: Endocrinopathy should be included in the differential diagnosis of altered mental status. This case additionally illustrates the challenges of managing adrenal insufficiency in the setting of hyperthyroidism and supports the use of thyroidectomy in this situation.

The More the Merrier: Integrating Multiple Models of Skeletal Maturity Improves the Accuracy of Growth Prediction.

BACKGROUND: Multiple systems using radiographic skeletal markers to measure development have been described, including the Greulich and Pyle Atlas (GP), the Fels Method (Fels), and the Sanders Hand Classification (Sanders). The purpose of this study was to quantitatively assess whether the integration of skeletal maturity assessment methods and demographic variables improves the accuracy of pediatric growth predictions over the use of skeletal markers or chronologic age alone. METHODS: The Brush Inquiry contains prospectively collected longitudinal data on children who lived in Cleveland, Ohio between 1926 and 1942. A total of 16 boys and 29 girls were selected for study. All had age, height, and an anteroposterior radiograph of the hand at each of 3 visits. Those visits occurred at 85%, 90%, and 95% of final height. We determined the growth completed at each visit by dividing the height observed by the final height at skeletal maturity. Boys and girls were analyzed separately using chronologic age, height, GP, Fels, and Sanders. The residual difference between the height predicted and actual height, as well as the SD of the prediction error of the cohort at each time point was calculated. To account for multiple visits from each subject, all linear models were produced using the generalized estimating equations (GEEs) procedure. RESULTS: For boys, age, GP, and Fels performed similarly in predicting growth remaining at all 3 time points. For girls, age, GP, and Fels performed similarly in predicting growth remaining at the 85% and 95% time points; however, the Fels Method demonstrated improved performance at the 90% time point compared with chronologic age (P = 0.0076) and GP alone (P = 0.0155). For both boys and girls, the most accurate multivariate GEE model with the lowest SD of prediction error integrated Fels, age, GP, Sanders, and height. CONCLUSIONS: The most accurate multivariate GEE model of growth prediction for both boys and girls integrated Fels, age, GP, Sanders, and height. When calculating the amount of growth remaining, it is prudent to integrate multiple systems for greater predictive accuracy. LEVEL OF EVIDENCE: Level III.

Bone Health in Childhood Chronic Disease.

Many children with chronic disease are now surviving into adulthood. As a result, there is a growing interest in optimizing bone health early in the disease course with the dual goals of improving quality of life during childhood and reducing life-long fracture risk. Risk factors for impaired bone health in these children include immobility, nutritional deficiency, exposure to bone toxic therapies, hormonal deficiencies affecting growth and pubertal development, and chronic inflammation. This review focuses on the chronic diseases of childhood most commonly associated with impaired bone health. Recent research findings and clinical practice recommendations, when available, for specific disorders are summarized.

Validation of a description of sarcopenic obesity defined as excess adiposity and low lean mass relative to adiposity.

BACKGROUND: This study aims to assess the construct validity of a body composition-defined definition of sarcopenic obesity based on low appendicular lean mass relative to fat mass (ALMIFMI ) and high fat mass index (FMI) and to compare with an alternative definition using appendicular lean mass index (ALMI) and percent body fat (%BF). METHODS: This is a secondary analysis of two cohort studies: the National Health and Examination Survey (NHANES) and the Health, Aging, and Body Composition study (Health ABC). Sarcopenic obesity was defined as low ALMIFMI combined with high FMI and was compared with a widely used definition based on ALMI and %BF cut-points. Body composition Z-scores, self-reported disability, physical functioning, and incident disability were compared across body composition categories using linear and logistic regression and Cox proportional hazards models. RESULTS: Among 14, 850 participants from NHANES, patients with sarcopenic obesity defined by low ALMIFMI and high FMI (ALMIFMI -FMI) had above-average FMI Z-scores [mean (standard deviation): 1.00 (0.72)]. In contrast, those with sarcopenic obesity based on low ALMI and high %BF (ALMI-%BF) had below-average FMI Z-scores. A similar pattern was observed for 2846 participants from Health ABC. Participants with sarcopenic obesity based on ALMIFMI -FMI had a greater number of disabilities, worse physical function, and a greater risk of incident disability compared with those defined based on ALMI-%BF. CONCLUSIONS: Body composition-defined measures of sarcopenic obesity defined as excess adiposity and lower-than-expected ALMI relative to FMI are associated with functional deficits and incident disability and overcome the limitations of using %BF in estimating obesity in this context.

Long-Term Follow-up of Hypophosphatemic Bone Disease Associated With Elemental Formula Use: Sustained Correction of Bone Disease After Formula Change or Phosphate Supplementation.

In this article, we describe the long-term outcomes of children who were previously reported to have developed hypophosphatemic bone disease in association with elemental formula use. An extended chart review allowed for an updated report of 34 children with regard to severity/duration of bone disease, extent of recovery, and time to correction using radiology reports and biochemical data. After implementation of formula change and/or phosphate supplementation, we found that serum phosphorus concentration increased and serum alkaline phosphatase activity decreased in all patients, normalizing by 6.6 ± 4.0 (mean ± SD) months following diagnosis. The decrease in serum alkaline phosphatase from diagnosis to the time of correction was moderately correlated with the concurrent increase in serum phosphorus (R = 0.48, P < .05). Age at diagnosis significantly correlated with time to resolution (R = 0.51, P = .01). This study supports the earlier report that bone disease associated with hypophosphatemia during elemental formula use responds to formula change and/or phosphate supplementation.

A 235 Kb deletion at 17q21.33 encompassing the COL1A1, and two additional secondary copy number variants in an infant with type I osteogenesis imperfecta: A rare case report.

BACKGROUND: Osteogenesis imperfecta (OI) is a rare group of disorders characterized by increased susceptibility to fractures due to genetically determined bone fragility. About 90% of cases are due to mutations in COL1A1 (17q21.33) or COL1A2 (7q21.3) resulting in quantitative or qualitative defects in type I collagen, a key structural constituent of bone. OI due to complete COL1A1 deletion is rare. METHODS: We present a case of OI type I in a Caucasian female referred at 10 months of age for investigation of multiple fractures associated with minimal or no known trauma, small stature, and blue sclera. Her father has four to five lifetime fractures, blue sclera, normal stature, and a 14.5 kilobase (kb) deletion of COL1A1 detected by targeted array performed at an outside institution. Microarray comparative genomic hybridization was performed on the proband and all members of the family. RESULTS: A previously unreported 235 kb deletion at 17q21.33 encompassing COL1A1, ITGA3, PDK2, SGCA, and HILS1 was detected in the proband. Also identified in both the proband and sibling is a maternally inherited 283 kb gain at 8p21.3 encompassing CSGALNACT1 and a 163 kb loss at 10q21.3 encompassing CTNNA3. Analysis in the father revealed the same size deletion at 17q21.33 as in the proband. CONCLUSION: Together with previously reported cases of COL1A1 deletions, this case report emphasizes the importance of a whole-genome DNA copy number assessment in patients suspected for OI, which will elucidate the presence of precise COL1A1 deletions and any pathogenic secondary copy number variations.