Borderline HbA2 levels: Dilemma in diagnosis of beta-thalassemia carriers.

There is inconsistency in the exact definition of diagnostic levels of HbA2 for ß thalassemia trait. While many laboratories consider HbA2 ≥4.0 % diagnostic, still others consider HbA2 ≥3.3 % or HbA2 ≥3.5 % as the cut-off for establishing ß thalassemia carrier diagnosis. This is because, over the years, studies have described ß thalassemia carriers showing HbA2 levels that lie above the normal range of HbA2 but below the typical carrier range of ß thalassemia. These, "borderline HbA2 levels", though not detrimental to health, are significant in ß thalassemia carrier diagnosis because they can lead to misinterpretation of results. In this review, we have evaluated the prevalence of borderline HbA2 levels and discussed the causes of borderline HbA2 values. We have also compiled an extensive catalogue of ß globin gene defects associated with borderline HbA2 levels and have discussed strategies to avoid misdiagnosing borderline HbA2 ß thalassemia carriers. Our analysis of studies that have delineated the cause of borderline HbA2 levels in different populations shows that 35.4 % [626/1766] of all individuals with borderline HbA2 levels carry a molecular defect. Among the positive samples, 17 % [299/1766] show ß globin gene defects, 7.7 % [137/1766] show α thalassemia defects, 2.7 % [49/1766] show KLF1 gene mutations, 2.3 % [41/1766] show the co-inheritance of ß and α thalassemia, 2.0 % [37/1766] show the co-inheritance of ß and δ thalassemia and 1.8 % [32/1766] show α globin gene triplication. It appears that a comprehensive molecular work up of the ß globin gene is the only definite method to detect borderline HbA2 ß thalassemia carriers, especially in populations with a high prevalence of the disease. The presence of associated genetic or acquired determinants may subsequently be assessed to identify the cause of borderline HbA2.

Challenging the dogma of the healthy heterozygote: Implications for newborn screening policies and practices.

Heterozygous (carrier) status for an autosomal recessive condition is traditionally considered to lack significance for an individual's health, but this assumption has been challenged by a growing body of evidence. Carriers of several autosomal recessive disorders and some X-linked diseases are potentially at risk for the pathology manifest in homozygotes. This minireview provides an overview of the literature regarding health risks to carriers of two common autosomal recessive conditions on the Recommended Uniform Screening Panel: sickle cell disease [sickle cell trait (SCT)] and cystic fibrosis (CF). We also consider and comment on bioethical and policy implications for newborn blood screening (NBS). Health risks for heterozygotes, while relatively low for individuals, are often influenced by intrinsic (e.g., other genomic variants or co-morbidities) and extrinsic (environmental) factors, which present opportunities for personalized genomic medicine and risk counseling. They create a special challenge, however, for developing screening/follow-up policies and for genetic counseling, particularly after identification and reporting of heterozygote status through NBS. Although more research is needed, this minireview of the SCT and CF literature to date leads us to propose that blanket terms such as "healthy heterozygotes" or "unaffected carriers" should be superseded in communications about NBS results, in favor of a more nuanced paradigm of setting expectations for health outcomes with "genotype-to-risk." In the molecular era of NBS, it remains clear that public health needs to become better prepared for the full range of applied genetics.

Cascade health service use in family members following genetic testing in children: a scoping literature review.

Cascade genetic testing is the identification of individuals at risk for a hereditary condition by genetic testing in relatives of people known to possess particular genetic variants. Cascade testing has health system implications, however cascade costs and health effects are not considered in health technology assessments (HTAs) that focus on costs and health consequences in individual patients. Cascade health service use must be better understood to be incorporated in HTA of emerging genetic tests for children. The purpose of this review was to characterise published research related to patterns and costs of cascade health service use by relatives of children with any condition diagnosed through genetic testing. To this end, a scoping literature review was conducted. Citation databases were searched for English-language papers reporting uptake, costs, downstream health service use, or cost-effectiveness of cascade investigations of relatives of children who receive a genetic diagnosis. Included publications were critically appraised, and findings were synthesised. Twenty publications were included. Sixteen had a paediatric proband population; four had a combined paediatric and adult proband population. Uptake of cascade testing varied across diseases, from 37% for cystic fibrosis, 39% to 65% for hypertrophic cardiomyopathy, and 90% for rare monogenic conditions. Two studies evaluated costs. It was concluded that cascade testing in the child-to-parent direction has been reported in a variety of diseases, and that understanding the scope of cascade testing will aid in the design and conduct of HTA of emerging genetic technologies to better inform funding and policy decisions.

The cost-effectiveness of genotyping versus sequencing for prenatal cystic fibrosis carrier screening.

OBJECTIVE: We investigated the cost-effectiveness of three sequential prenatal cystic fibrosis (CF) carrier screening strategies: genotyping both partners, genotyping one partner then sequencing the second, and sequencing both partners. METHOD: A decision-analytic model compared the strategies in a theoretical cohort of four million pregnant couples in the US population and five racial/ethnic sub-populations. Inputs were obtained from literature and varied in sensitivity analysis. Outcomes included cost per quality-adjusted life year (QALY), missed carrier couples, affected newborns, missed prenatal diagnoses, terminations, and procedure-related losses. The cost-effectiveness threshold was $100,000/QALY. RESULTS: Sequencing both partners identified 1099 carrier couples that were missed by genotyping both partners, leading to 273 fewer missed prenatal diagnoses, 152 more terminations, and 152 fewer affected newborns. A similar trend was observed in the genotyping followed by sequencing strategy. The incremental cost-effectiveness ratio of genotyping followed by sequencing compared to genotyping both partners was $180,004/QALY and the incremental cost-effectiveness ratio of sequencing both partners compared to genotyping followed by sequencing was $17.6 million/QALY. Sequencing both partners was cost-effective below $339 per test, genotyping/sequencing between $340 and $1837, and genotyping both partners above $1838. Sequencing was not cost-effective among five racial/ethnic sub-populations. CONCLUSION: Despite improved outcomes, sequencing for prenatal CF carrier screening was not cost-effective compared to genotyping. The clinical significance of the incremental cost-effectiveness of CF carrier screening is a matter of deliberation for public policy debate.

Recommendation of premarital genetic screening in the Syrian Jewish community based on mutation carrier frequencies within Syrian Jewish cohorts.

BACKGROUND: There is a paucity of information available regarding the carrier frequency for autosomal recessive pathogenic variants among Syrian Jews. This report provides data to support carrier screening for a group of autosomal recessive conditions among Syrian Jews based on the population frequency of 40 different pathogenic variants in a cohort of over 3800 individuals with Syrian Jewish ancestry. METHODS: High throughput PCR amplicon sequencing was used to genotype 40 disease-causing variants in 3840 and 5279 individuals of Syrian and Iranian Jewish ancestry, respectively. These data were compared with Ashkenazi Jewish carrier frequencies for the same variants, based on roughly 370,000 Ashkenazi Jewish individuals in the Dor Yeshorim database. RESULTS: Carrier screening identified pathogenic variants shared among Syrian, Iranian, and Ashkenazi Jewish groups. In addition, alleles unique to each group were identified. Importantly, 8.2% of 3401 individuals of mixed Syrian Jewish ancestry were carriers for at least one pathogenic variant. CONCLUSION: The findings of this study support the clinical usefulness of premarital genetic screening for individuals with Syrian Jewish ancestry to reduce the incidence of autosomal recessive disease among persons with Syrian Jewish heritage.

What women want: General population perspectives and access to preconception expanded carrier screening.

OBJECTIVE: Expanded carrier screening (ECS) assesses the risk of individuals and couples of having a child affected with a set of genetic conditions. Carriers have options available to optimize pregnancy outcomes based on personal values and preferences. The greatest range of options is available prior to pregnancy, therefore professional societies recommend this screening be performed preconception. This study aimed to assess awareness of, and interest in, ECS in women preconception. Additionally, it aimed to evaluate preferences for timing and location of education and availability of ECS. METHODS: A total of 260 nulliparous women from the general population were surveyed through Qualtrics, a national market research survey platform. Data were delineated using descriptive statistics. RESULTS: Of this cohort, 43.5% reported being aware of ECS prior to the survey and 77.8% indicated interest. Those previously aware were first informed by family, friends, or independent online research. Interest was primarily driven by a desire for reassurance and to make informed decisions about future pregnancies. Interested respondents indicated a willingness to request testing from providers. Participants showed a preference for education and access from a healthcare provider in person. CONCLUSION: These findings provide insight regarding when and where to best educate and reach women prior to pregnancy about ECS to maximize pregnancy outcomes.

Implementation of fragile X syndrome carrier screening during prenatal diagnosis: A pilot study at a single center.

BACKGROUND: Fragile X syndrome (FXS) is the most common inherited form of intellectual disability. Prenatal screening of FXS allows for early identification and intervention. The present study explored the feasibility of FXS carrier screening during prenatal diagnosis for those who were not offered screening early in pregnancy or prior to conception. METHODS: Pregnant women to be offered amniotic fluid testing were recruited for the free voluntary carrier screening at a single center between August, 2017 and September, 2019. The number of CGG repeats in the 5' un-translated region of the fragile X mental retardation gene 1 (FMR1) was determined. RESULTS: 4286 of 7000 (61.2%) pregnant women volunteered for the screening. Forty (0.93%), five (0.11%), and three (0.07%) carriers for intermediate mutation (45-54 repeats), premutation (55-200 repeats) and full mutation (>200 repeats) of the FMR1 gene were identified respectively. None of the detected premutation alleles were inherited by the fetuses. Of the three full mutation carrier mothers, all had a family history and one transmitted a full mutation allele to her male fetus. CONCLUSION: Implementation of FXS carrier screening during prenatal diagnosis may be considered for the need to increase screening for FXS.

International consensus on initial screening and follow-up of asymptomatic SDHx mutation carriers.

Approximately 20% of patients diagnosed with a phaeochromocytoma or paraganglioma carry a germline mutation in one of the succinate dehydrogenase (SDHx) genes (SDHA, SDHB, SDHC and SDHD), which encode the four subunits of the SDH enzyme. When a pathogenic SDHx mutation is identified in an affected patient, genetic counselling is proposed for first-degree relatives. Optimal initial evaluation and follow-up of people who are asymptomatic but might carry SDHx mutations have not yet been agreed. Thus, we established an international consensus algorithm of clinical, biochemical and imaging screening at diagnosis and during surveillance for both adults and children. An international panel of 29 experts from 12 countries was assembled, and the Delphi method was used to reach a consensus on 41 statements. This Consensus Statement covers a range of topics, including age of first genetic testing, appropriate biochemical and imaging tests for initial tumour screening and follow-up, screening for rare SDHx-related tumours and management of elderly people who have an SDHx mutation. This Consensus Statement focuses on the management of asymptomatic SDHx mutation carriers and provides clinicians with much-needed guidance. The standardization of practice will enable prospective studies in the near future.

The Molecular Bases Study of the Inherited Diseases for the Health Maintenance of the Beef Cattle.

The article highlighted the problem of meat cattle genetic defects. The aim was the development of DNA tests for some genetic defects diagnostics, the determination of the animal carriers and their frequencies tracking in time. The 1490 DNA samples from the Aberdeen Angus (n = 701), Hereford (n = 385), Simmental (n = 286) and Belgian Blue (n = 118) cattle have been genotyped on the genetic defects by newly created and earlier developed DNA tests based on AS-PCR and PCR-RFLP methods. The Aberdeen Angus cattle genotyping has revealed 2.38 ± 0.31% AMC-cows and 1.67 ± 0.19 % AMC-bulls, 0.65 ± 0.07% DDC-cows and 0.90 ± 0.10% DDC-bulls. The single animals among the Hereford cattle were carriers of MSUD and CWH (on 0.27 ± 0.05%), ICM and HY (on 0.16 ± 0.03%). The Simmental cattle were free from OS. All Belgian Blue livestock were M1- and 0.84%-CMD1-carriers. The different ages Aberdeen Angus cattle genotyping has shown the tendency of the AMC- and DDC frequencies to increase in the later generations. The statistically significant increase of DDC of 1.17% in the cows' population born in 2019 compared to those born in 2015 allows concluding the further development of the DNA analysis-based measures preventing the manifestation of the genetic anomalies in meat cattle herds is necessary.

Is it time to report carrier state for recessive disorders in every microarray analysis?-A pilot model based on hearing loss genes deletions.

This study aimed to examine the implications of reporting heterozygous losses of recessive genes in Chromosomal Microarray Analysis (CMA), based on the incidence of microdeletions of three common hearing impairment genes in the local cohort and the prevalence of sequence variants in these genes in worldwide databases. Prevalence of heterozygous microdeletions in OTOA and STRC genes, as well as deletions in the DFNB1 locus encompassing GJB6 gene, was determined using electronic database of Rabin Medical Center. ClinVar archive and Deafness Variation Database were used to generate a list of clinically significant sequence variants in these three genes, as well as GJB2 gene, and estimation of the frequency of sequence variants was performed. Of the 19,189 CMA tests were performed in our laboratory, 107 STRC microdeletions were found (0.56%), followed in frequency by OTOA deletions (39, 0.2%), and DFNB1 locus deletions (10, 0.05%). The estimated risk for a hearing loss in the examined individual carrying the microdeletion was estimated as 0.11-0.67% for STRC, 0.016-0.13% for OTOA, and 1.9-7.5% in the DFNB1 locus (including double heterozygocity with GJB2 clinically significant sequence variants). The risks were higher in specific populations. In conclusion, we believe that that general decision whether to report or to disregard such incidental findings cannot be part of a uniform policy, but rather based on a detailed evaluation of origin-specific variants for each gene, with a careful consideration and discussion whether to include the microdeletion in the final report for each patient.

Mainstream genetic testing for breast cancer patients: early experiences from the Parkville Familial Cancer Centre.

The demand for genetic testing of hereditary breast cancer genes such as BRCA1 and BRCA2 has continued to increase with the lowering costs of testing, raised awareness in the general public, and implications for breast cancer treatment when a patient is identified as having a germline pathogenic variant. Historically within Australia, patients affected by high genetic risk breast cancers have been referred to a familial cancer centre (FCC) for assessment and testing, resulting in wait times for an appointment for pre- and post-test genetic counselling and an increased demand on the public-funded FCC. To improve patient access and pace of genetic testing, as well as refocus FCC resources, a mainstream clinical genetic testing program was rolled out in September 2017 through the Parkville FCC (PFCC) in Australia at 10 hospital sites. This program enables specialist doctors of eligible patients affected by breast cancer to arrange genetic testing directly at an oncology/surgical appointment and follow up the results as part of the patients' routine clinical care. In this model, the specialist doctor is responsible for any treatment implications of the genetic test result, and the PFCC is responsible for result interpretation, future cancer risk, family cascade testing and segregation testing where warranted. To date the program has had successful uptake, a notable pathogenic variant detection rate, reduced the burden on the PFCC enabling a reallocation of resources and has streamlined the process of genetic testing for eligible patients. Investigation into the patient and clinician experiences of the mainstream program is required.

Health practitioners' perceptions of the barriers and enablers to the implementation of reproductive genetic carrier screening: A systematic review.

BACKGROUND: As interest in reproductive genetic carrier screening rises, with increased availability, the role of healthcare practitioners is central in guiding uptake aligned with a couples' values and beliefs. Therefore, practitioners' views on implementation are critical to the success of any reproductive genetic carrier screening programme. AIM: To explore healthcare practitioners' perceptions of the barriers and enablers to implementation. MATERIALS & METHODS: We undertook a systematic review of the literature searching seven databases using health practitioner, screening and implementation terms returning 490 articles. RESULTS: Screening led to the inclusion of 26 articles for full-text review. We found three interconnected themes relating to reproductive genetic carrier screening: (i) use and impact, (ii) practitioners' beliefs and expectations and (iii) resources. DISCUSSION: Barriers and enablers to implementation were present within each theme and grouping these determinants by (a) community for example lack of public interest, (b) practitioner for example lack of practitioner time and (c) organisation for example lack of effective metrics, reveals a preponderance of practitioner barriers and organisational enablers. Linking barriers with potential enablers leaves several barriers unresolved (e.g., costs for couples) implying additional interventions may be required. CONCLUSION: Future research should draw on the findings from this study to develop and test strategies to facilitate appropriate offering of reproductive genetic carrier screening by healthcare practitioners.

Parental exome analysis identifies shared carrier status for a second recessive disorder in couples with an affected child.

Consanguinity, commonplace in many regions around the globe, is associated with an increased risk of autosomal recessive (AR) genetic disorders. Consequently, consanguineous couples undergoing preimplantation genetic diagnosis (PGD) for one Mendelian disorder may be at increased risk for a child with a second, unrelated AR genetic disorder. We examined the yield of exome analysis for carrier screening of additional AR disorders, beyond the primary diagnosis, amongst consanguineous vs. non-consanguineous populations. Parental samples from trio exomes of 102 consanguineous families and 105 non-consanguineous controls were evaluated for shared carrier status, after disregarding the primary molecular diagnosis. Results were sub-classified according to disease severity. Secondary shared carrier status for pathogenic and likely pathogenic variants leading to AR disorders of moderate to profound severity was identified in 10/102 (9.8%) consanguineous couples, as compared to 1/105 (0.95%) non-consanguineous couples (χ2 = 8.0565, p value < 0.005). Higher inbreeding coefficient values, calculated from individual exomes, correlated with secondary shared carrier status for diseases of moderate to profound severity (r = 0.17, p value < 0.0125). Our results indicate that consanguineous couples undergoing PGD are at increased risk for a second genetic disease of moderate to profound severity. This study represents an underestimate of the rate of secondary shared carrier status due to inability to detect deep intronic variants, no assessment of copy number variants, and false negative results stemming from stringent variant interpretation. False positive results may result from inaccuracies in public databases. Additional studies in consanguineous populations will determine whether exome-based carrier screening should be recommended to all couples undergoing PGD.

Which types of conditions should be included in reproductive genetic carrier screening? Views of parents of children with a genetic condition.

Reproductive genetic carrier screening identifies couples with an increased chance of having children with autosomal and X-linked recessive conditions. Initially only offered for single conditions to people with a high priori risk, carrier screening is becoming increasingly offered to individuals/couples in the general population for a wider range of genetic conditions. Despite advances in genomic testing technology and greater availability of carrier screening panels, there is no consensus around which types of conditions to include in carrier screening panels. This study sought to identify which types of conditions parents of children with a genetic condition believe should be included in carrier screening. Participants (n = 150) were recruited through Royal Children's Hospital (RCH) Melbourne outpatient clinics, the Genetic Support Network of Victoria (GSNV) and a databank of children with hearing loss (VicCHILD). This study found that the majority of participants support offering carrier screening for: neuromuscular conditions (n = 128/134, 95.5%), early fatal neurodegenerative conditions (n = 130/141, 92.2%), chronic multi-system disorders (n = 124/135, 91.9%), conditions which cause intellectual disability (n = 128/139, 92.1%) and treatable metabolic conditions (n = 120/138, 87.0%). Views towards the inclusion of non-syndromic hearing loss (n = 88/135, 65.2%) and preventable adult-onset conditions (n = 75/135, 55.6%) were more mixed. Most participants indicated that they would use reproductive options to avoid having a child with the more clinically severe conditions, but most would not do so for clinically milder conditions. A recurring association was observed between participants' views towards carrier screening and their lived experience of having a child with a genetic condition.

Evaluation and classification of severity for 176 genes on an expanded carrier screening panel.

BACKGROUND: Disease severity is important when considering genes for inclusion on reproductive expanded carrier screening (ECS) panels. We applied a validated and previously published algorithm that classifies diseases into four severity categories (mild, moderate, severe, and profound) to 176 genes screened by ECS. Disease traits defining severity categories in the algorithm were then mapped to four severity-related ECS panel design criteria cited by the American College of Obstetricians and Gynecologists (ACOG). METHODS: Eight genetic counselors (GCs) and four medical geneticists (MDs) applied the severity algorithm to subsets of 176 genes. MDs and GCs then determined by group consensus how each of these disease traits mapped to ACOG severity criteria, enabling determination of the number of ACOG severity criteria met by each gene. RESULTS: Upon consensus GC and MD application of the severity algorithm, 68 (39%) genes were classified as profound, 71 (40%) as severe, 36 (20%) as moderate, and one (1%) as mild. After mapping of disease traits to ACOG severity criteria, 170 out of 176 genes (96.6%) were found to meet at least one of the four criteria, 129 genes (73.3%) met at least two, 73 genes (41.5%) met at least three, and 17 genes (9.7%) met all four. CONCLUSION: This study classified the severity of a large set of Mendelian genes by collaborative clinical expert application of a trait-based algorithm. Further, it operationalized difficult to interpret ACOG severity criteria via mapping of disease traits, thereby promoting consistency of ACOG criteria interpretation.

EMQN best practice guidelines for genetic testing in dystrophinopathies.

Dystrophinopathies are X-linked diseases, including Duchenne muscular dystrophy and Becker muscular dystrophy, due to DMD gene variants. In recent years, the application of new genetic technologies and the availability of new personalised drugs have influenced diagnostic genetic testing for dystrophinopathies. Therefore, these European best practice guidelines for genetic testing in dystrophinopathies have been produced to update previous guidelines published in 2010.These guidelines summarise current recommended technologies and methodologies for analysis of the DMD gene, including testing for deletions and duplications of one or more exons, small variant detection and RNA analysis. Genetic testing strategies for diagnosis, carrier testing and prenatal diagnosis (including non-invasive prenatal diagnosis) are then outlined. Guidelines for sequence variant annotation and interpretation are provided, followed by recommendations for reporting results of all categories of testing. Finally, atypical findings (such as non-contiguous deletions and dual DMD variants), implications for personalised medicine and clinical trials and incidental findings (identification of DMD gene variants in patients where a clinical diagnosis of dystrophinopathy has not been considered or suspected) are discussed.

How to manage BRCA mutation carriers?

Inherited mutations in BRCA1 and BRCA2 genes increase the risk of development of cancer in organs especially in breast and ovary. Prevention and screening in BRCA mutation carriers are of high importance. Prophylactic surgeries are possible but are still insufficiently performed because they require surgical procedures in healthy patients. Guidelines for the management of BRCA mutations carriers must absolutely be part of the standard practice of all those involved in the management of these patients to increase the impact of the implementation of these preventive measures. There is no screening recommended for ovarian cancer. A risk-reducing bilateral salpingo-oophorectomy should be performed from age 35 to 40 years for BRCA1 mutation carriers and 40 to 45 years for BRCA2 mutation carriers. A screening for breast cancer should be performed annually from 30 years old by breast MRI and mammography. A risk-reducing bilateral mastectomy is recommended with nipple sparing mastectomy and immediate breast reconstruction from 30 years and before 40 years. A multidisciplinary care must be implemented for these patients with an important psychological support.

Prenatal diagnosis of 2q13 duplications: The crucial role of the family survey in genetic counseling on novel copy number variations.

In recent years, the introduction of novel genome analysis technologies (such as array comparative genomic hybridization) has enabled the prenatal diagnosis of various recurrent copy number variations (CNVs). Some of these CNVs have been linked to a greater susceptibility of developmental and neuropsychiatric disorders; for example, recurrent duplication at the 2q13 locus is associated with developmental delay, dysmorphism and intellectual disability. However, this CNV has low penetrance and variable clinical expressivity. It also can be observed in healthy controls and can be transmitted by unaffected parents, making genetic counseling especially challenging. Here, we report on the inheritance of a 2q13 duplication in an asymptomatic family; the case highlights the role of the family survey in genetic counseling with regard to novel CNVs diagnosed before birth.

Experience with Parent Follow-Up for Communication Outcomes after Newborn Screening Identifies Carrier Status.

OBJECTIVE: To conduct interviews with a multiyear sample of parents of infants found to have heterozygous status for sickle cell hemoglobinopathy or cystic fibrosis during newborn blood screening (NBS). STUDY DESIGN: Interviewers with clinical backgrounds telephoned parents, and followed a structured script that blended follow-up and research purposes. Recruiting followed several steps to minimize recruiting bias as much as possible for a NBS study. RESULTS: Follow-up calls were conducted with parents of 426 infant carriers of sickle cell hemoglobinopathy, and 288 parents of cystic fibrosis carriers (34.8% and 49.6% of those eligible). Among these, 27.5% and 7.8% had no recollection of being informed of NBS results. Of those who recalled a provider explanation, 8.6% and 13.0% appraised the explanation negatively. Overall, 7.4% and 13.2% were dissatisfied with the experience of learning about the NSB result. Mean anxiety levels were low but higher in the sickle cell hemoglobinopathy group (P < .001). Misconceptions that the infant might get the disease were present in 27.5% and 7.8% of parents (despite zero actual risk for disease). Several of these data were significantly predicted by NBS result, health literacy, parental age, and race/ethnicity factors. CONCLUSIONS: Patient-centered public health follow-up can be effective after NBS identifies carrier status. Psychosocial complications were uncommon, but harms were substantial enough to justify mitigation.