Research questions

They were translated into PICO format (acronym for Population-Intervention-Comparator-Outcome) to guide the literature search. The main PICO question addressed was: What sort of endocrinologic follow-up is recommended to monitor the onset of endocrinologic diseases in NKX2-1-related disorders?. It was divided into three research questions to cover all the aspects related to the detection, diagnosis, treatment, and follow-up of endocrine alterations in NKX2-1-RD. An ongoing study will address treatment and follow-up.

In the present study, we focus on the research questions about detection and diagnosis: 1) What are the best procedures for initial screening of endocrine diseases in patients with NKX2-1-related disorders?, and 2) What are the best procedures for diagnosis of endocrine diseases in patients with NKX2-1-related disorders?. The protocols of the systematic reviews were previously registered in the International Prospective Register of Systematic Reviews (PROSPERO) repository with the identifications, CRD42022340996 and CRD42022341002, respectively.

Eligibility criteria

Specific inclusion criteria were employed to select the relevant studies for the systematic reviews. The population criteria were common for both research questions: patients of all ages with genetic confirmation of the disease (pathogenic variants in NKX2-1, previously known as TTF-1, or deletion in the 14q13.3 chromosome) with hypothyroidism. Non-human studies and patients without genetic confirmation of the disease were excluded. Given the rare condition, any comparator was used. The interventions and outcomes were specific to each research question:

1. What are the best procedures for initial screening of endocrine diseases in patients with NKX2-1-related disorders? Interventions: they were divided into antenatal screening (fetus ultrasound examination, cordocentesis, fetal blood sampling, and family inheritance of TTF-1/NKX2-1 mutation or deletion) and/or neonatal screening (thyroid hormone levels in blood spots and hearing tests). Outcomes: detection of suspicion of hypothyroidism, thyroid dyshormonogenesis and/or dysgenesis, neurodevelopmental alterations, and congenital malformations in the newborn.
2. What are the best procedures for the diagnosis of endocrine diseases in patients with NKX2-1-related disorders? Interventions: quantification of TSH and fT4 levels in serum and thyroid gland imaging tests (scintigraphy and/or ultrasonography). Outcomes: thyroid hormone levels in serum to confirm the presence of hypothyroidism and thyroid gland volume and location.

Interventions related to hypothyroidism but not to NKX2-1-RD were excluded. The included study designs were primary studies, systematic reviews, and randomized controlled trials (RCTs). Narrative reviews, protocols, conference articles, editorials, letters to the editor, and those whose full text was unavailable to be retrieved were excluded.

Search strategy

A systematic literature search was carried out for each research question to review the scientific evidence. PubMed, Embase, the Cochrane Library, and MEDLINE (Ovid) were searched as reference databases. Other resources were APA PsyncINFO, CINAHL Complete, the TRIP medical database, and ICTPR. Subject-specific databases were included to retrieve information in rare disease resources such as Orphanet, EURORDIS, NORD, RARE-Best Practices, and Gene Reviews. Finally, the International Health Technology Assessment (HTA) database, a systematic review-specific resource, was included for the retrieval of technology assessments.

The search covered the period from January 2002 to May 2022, and it was updated in June 2023. The included records date back to 2002, when pathogenic variants in the NKX2-1 gene were first associated with benign hereditary chorea [19]. Both controlled language (descriptors) and free terminology were used to search for studies. The initial strategies were carried out in MEDLINE (Ovid) and later adapted to each database’s syntax. The original search strategies are specified in S2 File.

Study screening and data extraction

The identified references were imported into the reference management section of the software application Covidence (https://www.covidence.org/), and the duplicates were removed. A different review was created for each question. Two authors independently filtered the references by title, abstract, and full text (BCH and CMG for the screening research question and BCH and JDOE for the diagnosis research question) according to the inclusion criteria. The disagreements between the reviewers were resolved by a third researcher, (JL). The data were extracted by two independent authors (BCH and JDOE) and recorded in Excel spreadsheets. Specific details of the studies (author, year of publication, study type, and location) and specific clinical data of each patient (age, sex, genetic tests, thyroid, neurological, and respiratory alterations, age at onset of hypothyroidism, screening and diagnosis methods, hormone levels, and thyroid gland volume and/or location alteration) were extracted.

Quality assessment

The quality of the studies was assessed independently by two authors (BCH and JDOE). Disagreements were resolved through discussion to reach a consensus. The quality assessment was carried out using specific tools for each type of study. The included references were classified as case reports, case series, and cohort studies. The tool developed by Murad et al. [20], used for case reports and case series, allows the evaluation of the methodological quality of studies with a low body of evidence but is essential for the decision-making process. It uses eight items categorized into four domains (selection, ascertainment, causality, and reporting). According to the criteria of the tool, the quality was rated by summing the scores of the eight binary responses (1 = low risk of bias, 0 = high risk of bias) into an aggregate score. The score ranges from 0 (poor quality) to 8 (high quality) [21]. The items of ascertainment, causality, and reporting were adapted to the specific clinical scenario included in the research questions. Regarding cohort studies, we used the Newcastle-Ottawa Scale (NOS), which can also be modified based on a special subject [22]. This tool uses three categories (selection, exposure, and comparability) with eight items to identify good-quality decisions using starts. One star can be given to each item in the selection (maximum of 4 stars) and outcome (maximum of 3 stars) categories, and a maximum of two stars for comparability. No studies were excluded based on low-quality grounds. The Grade of Recommendation, Assessment, Development, and Evaluation System (GRADE) was established to evaluate the quality of each outcome provided by the evidence of the studies. This system sets four categories (high, moderate, low, and very low) with five factors: risk of bias, inconsistency, indirectness, imprecision, and publication bias [23].

Study selection

During the initial search, a total of 1012 records were located. After removing duplicates, 987 potentially relevant studies remained (649 in the first research question and 338 in the second one). Title and abstract screening was carried out, yielding 106 and 50 studies, respectively. According to inclusion and exclusion criteria, 63 studies were included following full-text screening for both questions. After removing common studies between both research questions, 46 studies were finally analyzed for this study. The screening processes (PRISMA flowcharts) [24] are depicted in Fig 1. The included and excluded articles after full-text review, along with the reasons for their exclusion, are included in S3 File. The excluded studies were classified according to the first criterion that did not fit the PICO format.

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Fig 1. PRISMA flowchart.

Illustration of the PRISMA flowchart depicting the title-abstract and full-text screening of the articles related to the detection and diagnosis of endocrine diseases in patients with NKX2-1-RD.

https://doi.org/10.1371/journal.pone.0303880.g001

Study characteristics

52% of the included studies were case series (n = 24), 43% were case reports (n = 20), and 5% were cohorts (n = 2). Due to the rare clinical condition studied, no anticipated randomized controlled trial or systematic review was available. 63% of the data originated from Europe (n = 29), 22% from Asia (n = 10), 13% from America (n = 6), and 2% from Australia (n = 1) (Table 1).

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Table 1. Presents a comprehensive list of references that were included in this study for the purpose of investigating the initial screening (detection) and diagnosis of thyroid diseases in individuals affected by NKX2-1-RD.

The author, year of publication, location, and the number of patients included in each study for screening and/or diagnosis are specified.

https://doi.org/10.1371/journal.pone.0303880.t001

Patients demographics

Only patients diagnosed with NKX2-1-RD and hypothyroidism were considered (113 patients). At the time of the study, 1.7% of patients were neonates (≤ 28 days of life) (n = 2), 41.6% were children (≤12 years of age) (n = 47), 14.1% were adolescents (13–19 years of age) (n = 16) and 18.6% were adults (20–65 years old) (n = 21). In 23.9% of patients (n = 27) the age was not reported. The calculated mean age was 15.3 years (range 28 days to 64 years). The gender was quite equally distributed (54% were females) (n = 61). Regarding pathogenic NKX2-1 gene variants or deletions, there were eight cases (7%) with splicing variants, thirty-three individuals (29%) with missense variants, fifty-three (47%) patients with frameshift variants, eighteen cases (16%) with large deletions, and one patient with an insertion of a 46-bp Alu sequence in the NKX2-1 gene [4]. One patient carried a double homozygous variant [25] and another two deletions in the short and long arms of chromosome 14 [26]. A patient carried a frameshift variant and a splicing variant [27]. The main technique used to identify pathogenic variants was sequencing in 71.7% of the patients (n = 81). Comparative genomic hybridization (CGH) was used in 10.6% of the cases (n = 12), while Fluorescence in situ Hybridization (FISH) and bacterial artificial chromosomes (BAC) were used in 1.8% of the cases each (n = 2). Next generation sequencing techniques were used in 7% (n = 8), and no diagnostic technique was reported in an additional 7% of patients. In 44 (38.9%) patients, the pathogenic variants were de novo. In 37 cases (32.7%), the type of inheritance was not provided.

In our study, CH was the most common endocrine alteration confirmed in 51 patients (45.1%). Compensated or subclinical hypothyroidism was present in twenty-three patients (20.3%), and gestational hypothyroidism during pregnancy was present in three patients (2.7%) [28–30]. The remaining 36 patients in our cohort of 113 presented overt hypothyroidism (31.9%). In addition to hypothyroidism, the presence of other endocrine alterations was found among the patients with NKX2-1-RD. A neonatal patient diagnosed with CH demonstrated multiple pituitary hormone deficiencies, including growth hormone and cortisol deficiency. The gonadotrophins were undetectable, and the pituitary gland was small with a normal stalk. Transient hyperinsulinism was present during the neonatal period, requiring a brief period of treatment [26]. The growth hormone deficiency and visual impairment were present in another patient diagnosed with CH [31], and in a patient with subclinical hypothyroidism who showed serum insulin-like growth factor-I deficiency too [32]. Immunodeficiency was significant in a patient diagnosed with primary hypothyroidism in the first year of age [33] and in another patient with subclinical hypothyroidism, fetal growth, a reduction of CD3/CD8, and an increase of the CD4/CD8 ratio [34]. An adult patient was diagnosed with hypothyroidism and another patient with CH during childhood [2], both of them with pituitary disfunction. The low levels of testosterone and luteinizing hormone resulted in hypogonadism that required treatment [35]. Two patients from the same study, diagnosed with mild hypothyroidism at an advanced age, showed a slightly low prolactin level and cystic pituitary mass [36].

Regarding clinical alterations related to NKX2-1-RD, 57.8% of patients reported neurological, thyroid, and lung disease (a complete triad). 29.3% of the patients had endocrine and neurological alterations, while only 10% had endocrine and respiratory involvement. Chorea was the most prevalent neurological disorder (88.6% of patients with neurological involvement), followed by gait disorders and neurodevelopmental delay. Neonatal respiratory distress was the most frequent manifestation (45%) in patients with respiratory alterations, followed by respiratory infections and diminished lung capacity. As respiratory alterations are common in premature births, we analyzed whether the presence of these alterations among the patients in our study could be due to early birth or to NKX2-1-RD. 62.8% of patients in this study reported respiratory alterations (n = 71), while only 4.2% of them (n = 3) were premature at birth [37, 38]. More details are presented in S4 File.

Initial screening strategies of endocrine alterations in patients with NKX2-1-RD

NBS were conducted utilizing blood spots to measure TSH and fT4 or T4 levels in the first days of life after birth. In this study, the results of the neonatal screening were reported in 21.2% of patients (n = 24) indicating CH in 16.8% (n = 15) of them. The mean and median blood TSH levels were 58.7 mU/L and 44 mU/L, respectively, reported in the 12.4% of patients (n = 14) with neonatal screening. In 9.7% of patients (n = 11), the blood TSH value was reported as normal or high but lacked numerical details. A detail to highlight is that Gillett et al. 2013 [39] reported markedly elevated TSH levels.

No consensus on hormone values for CH detection was found, making it difficult to establish a standard range. Some patients had hormone levels above the normal range, indicating endocrine alteration, but no numbers were given. Eight studies (17.4%) reported a TSH threshold of 10 mU/L or less for neonatal screening to consider CH. Two studies set the threshold to 16.9 mU/L [28, 40]. Other studies did not report cut-off values. All CH confirmatory cases had TSH values above thresholds. Only one study reported fT4 in neonatal screening and TSH in CH patients [41]. Four studies measured T4 [28, 40, 42, 43]. The mean and median (n = 3) were 124.8nmol/L and 151.2 nmol/L. These values were considered normal or low regarding the threshold established by each study. S5 File shows neonatal thyroid hormone levels.

Diagnosis strategies of endocrine alterations in patients with NKX2-1-RD

Diagnostic tests measured the levels of TSH and fT4 or T4 in serum to confirm the presence of hypothyroidism after a previous screening test or to diagnose for the first time a thyroid alteration. 34.5% of patients (n = 39) were diagnosed at neonatal age within the first month of life, while 41.6% (n = 47) of the patients were diagnosed later in life. The mean age for diagnosis was 15.1 years (range 10 months to 60 years). In the remaining patients, no available data on age at diagnosis was reported. 42.5% of patients (n = 48) were positive for CH. The mean and median values of TSH levels in serum were 86.1mU/L and 29.2 mU/L, respectively. In 14.2% of patients (n = 16) with a diagnosis test, no numerical data was specified, only indications of normal or high TSH levels. One patient showed a high level of TSH, the same patient with elevated TSH at the neonatal screening described previously [39]. As neonatal screening tests, there was no clear consensus on the threshold to establish the TSH levels in serum in diagnosis to confirm hypothyroidism for patients with NKX2-1-RD. The cut-off value of TSH in serum was set to a maximum of 6.3 mU/L in 14 studies (30.4%). In one study, the threshold was set at 0.6 mU/L [44] and in the other three studies, the threshold values reached a maximum of 13.1 mU/L [45], 15 mU/L [29] or 24 mU/L [46]. Regarding fT4 levels in serum, they were reported in 24.8% of patients (n = 28) from 19 studies (41.3%). The mean value was 11.6 pmol/L and the median was 12.4 pmol/L. The minimum and maximum thresholds to detect altered fT4 values showed variability among studies. T4 levels were reported in 9 patients from 3 studies [5, 42, 47]. The mean and median values were 72.7 nmol/L and 75.9 nmol/L, respectively. Details about diagnosis tests and hormone levels are presented in S5 File.

Complementary tests for endocrine, neurological and respiratory alterations in patients with NKX2-1-RD

Scintigraphy and ultrasonography were the most frequently used techniques to evaluate the etiology (dysgenesis or gland in situ) of hypothyroidism through the size, location, and echostructure of the thyroid gland, along with the diagnostic tests. Thyroid ultrasonography was used in 33 patients (29.2%) and scintigraphy in 25 (22.1%). It was reported that 32 patients (28.3%) had abnormalities in their thyroid gland: 16.8% of patients (n = 19) had hypoplasia, 4.4% (n = 5) had an ectopic thyroid gland, 3.5% (n = 4) had a thyroid gland with altered uptake, three of them with low uptake and one with no uptake in the neck, 1 patient had hemiagenesis, and 1 had athyreosis. The defect in a patient was not specified [29]. A hypoechogenic nodule was observed in another patient [48]. Perchlorate discharge test and a hearing test were carried out in a study, but no hearing loss was detected, and the thyroid was considered normal [49]. Fetus ultrasound, cordocentesis, and fetal blood sampling were included in the research question as interventions for neonatal screening to cover all the possible evidence about detection procedures in patients with NKX2-1-RD. No evidence was finally found in this review about these interventions. Details about thyroid pathology are presented in S5 File.

Regarding neurological abnormalities, 23% of patients underwent additional tests such as MRI, SPET, and SPECT [36, 50–52]. The Denver developmental test, the McCarthy cognitive test, and the WAIS-III scale were utilized for psychomotor, cognitive, and intelligence evaluations, respectively [28, 41]. These tests were conducted during the first 2–3 years of life, when the first symptoms of chorea appeared. Tests for respiratory function were reported in 12.3% of patients (cardiac ultrasound, lung tomography, lung biopsies, and respiratory function tests, among others). These tests were commonly conducted from birth when infants displayed neonatal respiratory distress [13, 33, 39, 53–55].

Quality assessment of the included studies

According to the assessment tools used in our systematic review, 72.7% (n = 32) of case series and case reports had poor methodological quality, 20.5% (n = 9) had medium quality, and 6.8% (n = 3) had good quality. One cohort study had poor methodological quality [56], whereas the second one had good quality [57]. The scores obtained for each item, as well as the total score for each reference, are shown in S6 File.

Quality assessment of the evidence

The fact that the available evidence came from case series or case reports downgrades the initial quality. Due to the lack of reported data and inadequate study design, it has not been possible to quantitatively estimate the risk of bias, inconsistency, indirectness, imprecision, and publication bias. The wide variability was due to the lack of concordance among studies in the threshold values of hormone levels and in the age at diagnosis of hypothyroidism. The indirectness of the study was considered low since the available evidence answered the initial research questions. Otherwise, the small sample size and the lack of reporting of interest outcomes lead to imprecision in the results.

In terms of publication bias, conducting an objective assessment was challenging due to the limited availability of data from unpublished studies. However, the reported results consistently align in terms of the detection and diagnosis strategies as well as hormone levels across all patients. The bibliographic searches were designed to be sensitive with the aim of retrieving all the related studies to the criteria of the research questions, but there is the possibility that some of them were lost. The overall quality of the evidence analyzed in this study is considered low due to the previously discussed factors.

Keys for screening and diagnosis of thyroid disorders in patients with NKX2-1-RD

1. Standardize the cut-off threshold for TSH in neonatal screening. Achieving a clear consensus on the TSH cut-off thresholds will facilitate early detection and prompt further evaluation, ensuring timely diagnosis. Further studies are needed to determine whether the current standard TSH level on blood filter paper at neonatal screening should be modified.
2. Expansion by healthcare providers of the screening and diagnostic practices for hypothyroidism even after the systematic neonatal screening during infancy, childhood, and in women affected by NKX2-1-RD to include the gestational period. A comprehensive assessment should be conducted in pregnant women with transient hypothyroidism, specifically looking for other manifestations that could be indicative of an underlying NKX2-1-RD.
3. Spread endocrine screening beyond thyroid screening in patients with NKX2-1-RD. Healthcare providers should consider including assessments of clinical manifestations for other endocrine deficiencies, allowing the determination of hormonal impairment. In cases of pituitary hormone deficiency, a systematic cerebral MRI should be performed to look after the size and structure of the pituitary gland.
4. Facilitate genetic testing. Given the genetic basis of NKX2-1-RD, consider incorporating genetic testing, such as NKX2-1 gene sequencing, WES, or Array CGH, into the diagnostic workup of patients with CH, particularly those with associated respiratory distress and/or neurological impairment and/or pituitary deficiency. Genetic testing can aid in the identification of specific gene variants or deletions associated with the disorder, thereby facilitating an accurate diagnosis and family counseling.
5. Improve neurological and respiratory assessment. In patients with NKX2-1-RD, conduct additional tests to assess neurological and respiratory abnormalities, such as MRI, SPET, SPECT, psychomotor, cognitive, and intelligence evaluations. Comprehensive evaluations can aid in the early detection and management of these symptoms, improving patient care.