You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


Introduction
============

Deficiencies in gonadotropins and sex hormones are detrimental for various stages of sexual maturation and lead to hypogonadotropic hypogonadism (HH).[@b1-tacg-7-177] The isolated form of HH is rare; however, its association with certain olfactory deficits (anosmia/hyposmia) that are part of Kallmann syndrome (KS) -- OMIM 300836 -- makes it more common.

KS can occur sporadically or be hereditary, and its incidence is 1:8,000 in males and 1:40,000 in females.[@b2-tacg-7-177],[@b3-tacg-7-177] Hereditary cases have been well documented but sporadic cases are more common, which suggests that de novo mutations are an important cause.[@b4-tacg-7-177]--[@b6-tacg-7-177]

There are at least six well-known genes involved in the etiology of KS: the *KAL1* gene, which encodes the anosmin-1 glycoprotein; the *FGFR1* (*KAL2*) gene, which encodes fibroblast growth factor receptor 1; the *PROKR2* (*KAL3*) and *PROK2* (*KAL4*) genes, which encode the prokineticin-2 receptor and prokineticin-2, respectively; and the *CDH7* (*KAL5*) and *FGF8* (*KAL6*) genes. Other genes have been reported to be associated with KS: *NELF, WDR11, HS6ST1, SEMA3A, HESX1*.[@b7-tacg-7-177],[@b8-tacg-7-177]

X-linked KS (X-KS) typically involves mutations in the *KAL1* gene, which is found on chromosome Xq22.3; such mutations can alter the form and function of the anosmin-1 protein, which is involved in the migration of olfactory neurons and neurons that produce gonadotropin-releasing hormone. Without anosmin-1, migration is impaired, which affects the patient's olfaction and gonadotropic hormone production.[@b6-tacg-7-177],[@b9-tacg-7-177],[@b10-tacg-7-177]

In combination with the profile of HH and anosmia/hyposmia, a patient may present with various other alterations; the following are some common findings: unilateral renal agenesis; cavus foot; cleft lip and palate; synkinesis; and auditory, visual or other sensory disturbances.[@b7-tacg-7-177] In any case, great phenotypic heterogeneity has been demonstrated in various studies, especially those of Quinton et al,[@b11-tacg-7-177] Oliveira et al[@b12-tacg-7-177] and Hardelin and Dodé,[@b13-tacg-7-177] suggesting that the activity of epigenetic factors or modifying genes COULD determine phenotypic variability.

Detailed clinical assessment of patients who are affected by HH and have anosmia/hyposmia has led to the discovery of other significant changes in this group, and adding genetic analysis permits appropriate genetic counseling and helps molecular understanding of KS.

Materials and methods
=====================

Ethical issues
--------------

Ethical consent was obtained according to the Declaration of Helsinki. Ethical approval was obtained from the Brazilian National Committee on Research Ethics (CEP/HUJBB -- Protocol Number 142/2008). All of the patients assessed agreed to participate in the study by signing an informed consent form.

Description of the samples
--------------------------

A clinical and molecular study was conducted with four KS patients and 12 of their family members, for a total of 16 individuals. The clinical study of those affected assessed the following parameters: 1) pattern of family inheritance; 2) stage of sexual maturation; 3) anthropometric data; 4) hormone levels (follicle-stimulating hormone \[FSH\], luteinizing hormone \[LH\], testosterone, estradiol, thyroid-stimulating hormone \[TSH\], free T4); 5) other associated conditions including cryptorchidism, renal malformation, synkinesis, and others; and 6) molecular analysis of the *KAL1* gene (NM_000216.2).

The family members underwent an assessment focused on the investigation of anosmia, identification of associated malformations and genetic study.

The olfactory assessment was conducted using the semiological test of the first cranial nerve based on the presentation of test tubes containing ten different common everyday odors to be identified by the patients and their family members. Chloroform was used to test for false positives because the olfactory nerve is not required to detect it.

Molecular analysis
------------------

DNA was extracted from peripheral blood samples taken from each individual by the phenol-chloroform method.[@b14-tacg-7-177] Exons 5, 6 and 9 of the *KAL1* gene (NM_000216.2) were selected for amplification by polymerase chain reaction (PCR) because they contained the greatest number of mutations described at the time of the present study.[@b15-tacg-7-177] The primers used for amplification reaction are shown in [Table S1](#SD1-tacg-7-177){ref-type="supplementary-material"}. The final reaction volume was 25 μL and included 100 ng of template DNA, 0.1 μM dNTPs, 0.1 μM of each primer, 1.5 μM MgCl 2, 1× PCR buffer (10 mM Tris-HCl, pH 8.4, and 50 mM KCl) and 1 U Platinum^®^ Taq DNA Polymerase (Invitrogen). After initial denaturing for 10 min at 95°C, the samples underwent 30 cycles under the following parameters: denaturing at 94°C for 1 minute, annealing at 54°C for 1 minute for exon 5, and at 56°C for 1 minute for exons 6 and 9, and extension at 72°C for 1 minute. The samples were checked by electrophoresis on a 1.5% agarose gel.

The sequencing reaction was performed using 1 μL of purified PCR product, 0.5 μL reverse primer, 2.0 μL of Big Dye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems^®^) and sterile deionized water to a final volume of 20 μL. The reaction was performed on a thermocycler with an initial denaturing at 96°C for 2 minutes, followed by 36 cycles of 96°C for 30 seconds, 54/56°C for 20 seconds (for exon 5, and exons 6 and 9, respectively), and 60°C for 4 minutes. The samples were treated and sent to an ABI 3110 sequencer for electrophoresis on 4.5% acrylamide gel (10 mL of 18.5 acrylamide:1 bis-acrylamide, 36 g of urea and 60 mL of 1× TBE). The sequencing results were interpreted using the ABI Analysis Software^®^.

Results
=======

Four male patients in the third decade of life presented with the clinical and laboratory profile of HH ([Table 1](#t1-tacg-7-177){ref-type="table"}) combined with anosmia confirmed by the semiological test. Four other features were observed that are common in KS ([Table 2](#t2-tacg-7-177){ref-type="table"}): unilateral renal agenesis, cryptorchidism, synkinesis and dental agenesis. Nystagmus, ptosis, auricular dysgenesis and cleft lip/palate were not found in any patient.

The molecular analysis detected the transversion of G to A at position 612 on exon 5 of the *KAL1* gene. This mutation was hemizygous in the four affected brothers. Five females heterozygous for this mutation were identified in the family, including the patients' mother ([Figure 1](#f1-tacg-7-177){ref-type="fig"}). The same mutation was not found in the remaining seven family members. This novel mutation generates a stop codon instead of a tryptophan amino acid at position 204 of the anosmin-1 protein.

Discussion
==========

KS is one of the most common causes of HH.[@b4-tacg-7-177] In male patients (who have a greater incidence) who present with a clear picture including delayed puberty and, therefore, likely hypogonadism, it is appropriate to question or investigate their olfactory function with possible investigation oriented toward KS. In the cases studied, the complaint of difficulty perceiving odors aided the clinical study.

X-linked inheritance is the best clinically characterized form of the disease.[@b11-tacg-7-177] Some associated conditions are more common in this type of inheritance, such as dental agenesis, synkinesis and renal agenesis.[@b16-tacg-7-177] All of these features were observed in the probands, reinforcing the suspicion for X-linked inheritance.

The cryptorchidism found in the four patients should be of high importance because it is often the most evident clinical feature, especially in pediatric patients when it is not possible to apply olfactory tests and hormone levels are of little value. In such cases, it is important to look for other signs such as the presence of micropenis. Additionally, a family history of the characteristic signs of KS should be investigated.[@b17-tacg-7-177]

Renal agenesis is present in 35%--40% of X-linked KS cases.[@b18-tacg-7-177] Other malformations of the urinary tract can also be found in such patients, including horseshoe kidney, kidney rotation, hydronephrosis, duplicated ureter, etc.[@b19-tacg-7-177] The importance of imaging exams, especially abdominal ultrasound, is clear because they can show renal abnormalities that are not clinically evident. Therefore, such exams should be considered imperative for KS patients.

Georgopoulos et al[@b20-tacg-7-177] and Sato et al[@b16-tacg-7-177] reported a greater incidence of right renal agenesis with a tendency for KS patients to present with this side affected, especially those with a *KAL1* mutation. Left renal agenesis is more commonly found in individuals without KS. The mechanisms that explain these findings are still unknown.

Synkinesis, which is considered the marker of X-linked inheritance and was present in the probands in this study, can be explained by an absence of inhibitory fibers that connect the two motor cortices, resulting in fewer fibers in the corpus callosum or even agenesis.[@b11-tacg-7-177] These alterations were not found in the studied patients with magnetic resonance imaging.

An analysis of the type of mutation that generates a protein truncated at codon 204 revealed interrupted synthesis at the first fibronectin type III-like domain. This finding agrees with the analysis by Georgopoulos et al,[@b20-tacg-7-177] who found many mutations in this domain related to renal alterations. A more recent study could not specifically attribute this renal phenotype to any of the genes known to cause the syndrome.[@b7-tacg-7-177]

Most cases require lifelong sex steroid hormone replacement to maintain sexual features and bone density, although there have been cases in which the profile of hypogonadism reversed after a period of hormone replacement[@b21-tacg-7-177] in patients who had not been genetically screened and in patients with detected mutations in the *KAL1*[@b22-tacg-7-177] and *KAL2/FGFR1* genes.[@b23-tacg-7-177]

Reversal of the disease is achieved when the individual maintains normal serum levels of sex hormones after a long period without hormone replacement, allowing the possibility of endogenous maintenance of complete virilization and fertility.[@b22-tacg-7-177] Although the estimated number of reversible cases is low, Quinton et al[@b21-tacg-7-177] have suggested that they are underestimated due to interruption of treatment by the patient, now eugonadal, without any effect on their clinical progression. Additionally, there may be a lack of follow-up by specialized professionals. Therefore, there may be reversible cases that remain unidentified.

Advances in the understanding of the genetic bases of KS have allowed increasingly complex molecular studies that can provide additional information on the pathogenesis of the disease and the genotype--phenotype associations.[@b7-tacg-7-177] Although it has been widely studied, novel recessive mutations of the *KAL1* gene are still being described in the literature, as in the present study and in a recent analysis of Chinese probands.[@b24-tacg-7-177]

In conclusion, the clinical and molecular studies conducted herein broadened the spectrum of the known pathogenic recessive mutations related to KS. The results will serve as the basis for appropriate genetic counseling to inform patients and their families about the disease and its repercussions, especially reproductive issues. Additionally, counselors can warn them about the risk of recurrence in the family and suggest early diagnosis of future patients so that treatment can be initiated at the ideal time.

Supplementary material
======================

###### 

Polymerase chain reaction primers used for amplification of *KAL1* exons 5, 6 and 9, their annealing temperature and product size

  Exon                                 Forward and reverse primers         Annealing temperature (°C)   Product size (bp)
  ------------------------------------ ----------------------------------- ---------------------------- -------------------
  5                                    5′ TTGTTTTAATTGATACGGTCCTGTTT 3′    54                           185
  5′ CAAGTTAATTTTTTGTGCGTAGCTATGT 3′                                                                    
  6                                    5′ CAACTAACATGTCGGAATAAAAAGTGA 3′   56                           130
  5′ TGAACATAGAGACAGTGAATCTGCATT 3′                                                                     
  9                                    5′ CATCTTGCCCAGGAATCTATAATTACT 3′   56                           147
  5′ TTGATACTGTGGCTTGACATTTACTTC 3′                                                                     

The authors thank all of the patients and the Endocrinology team from João de Barros Barreto University Hospital -- UFPA and the Genetics team from the Laboratory of Human and Medical Genetics -- UFPA. Financial support was received from UFPA(PROPESP)/FADESP and CAPES.

**Disclosure**

The authors have no conflicts of interest in this work.

![The inheritance pattern for the family studied shows the hemizygous affected individuals with Kallmann syndrome, and heterozygous carriers who were detected by genetic analysis (**A**). The chromatogram patterns found in the study are shown above with mutations indicated by an arrow (**B**).\
**Notes:** The generations in the family are given by I, II and III (**A**).](tacg-7-177Fig1){#f1-tacg-7-177}

###### 

Summary of laboratorial tests in the first assessment

                                  Patients                                    
  ------------------------------- ------------- -------------- -------------- ----
  FSH (1.24--7.8 IU/L)            0.7 IU/L      0.31 IU/L      0.73 IU/L      ND
  LH (6--23 IU/L)                 0.13 IU/L     0.10 IU/L      0.3 IU/L       ND
  Testosterone (438--697 ng/dL)   11.52 ng/dL   28.81 ng/dL    13.54 ng/dL    ND
  Estradiol (10--40 pg/mL)        8 pg/mL       2.7 pg/mL      35.4 pg/mL     ND
  TSH (0.4--4.0 μU/mL)            1.6 μU/mL     1.3 μU/mL      1.4 μU/mL      ND
  Free T4 (9.0--19.3 nmol/mL)     12 nmol/mL    12.5 nmol/mL   12.6 nmol/mL   ND
  Karyotype                       46, XY        46, XY         46, XY         ND

**Note:** Normal values are given in parentheses in the left column.

**Abbreviations:** ND, no data available; FSH, follicle-stimulating hormone; LH, luteinizing hormone; TSH, thyroid-stimulating hormone.

###### 

Summary of clinical findings in Kallmann syndrome patients

                                                                                                 Patients                                                                                                                                         
  ---------------------------------------------------------------------------------------------- ----------------------------------------------------------------------- ------------------------------------ ----------------------------------- ----------------------
  Sex                                                                                            Male                                                                    Male                                 Male                                Male
  Age (years)                                                                                    30                                                                      27                                   26                                  23
  Sense of smell                                                                                 Hyposmia                                                                Anosmia                              Anosmia                             Anosmia
  Sexual maturation[\*](#tfn3-tacg-7-177){ref-type="table-fn"} (Tanner stage)                    G4P4B3                                                                  G4P4B3                               G4P4B3                              G2P2B3
  Testicular volume and cryptorchidism[\*\*](#tfn4-tacg-7-177){ref-type="table-fn"} right/left   C-R/3 mL                                                                4 mL/C-L                             C-R/4 mL                            C-R/C-L
  Renal malformation                                                                             Right kidney agenesis                                                   Left kidney agenesis                 Right kidney agenesis               Left kidney agenesis
  Dental agenesis                                                                                First, second and third lower right molars and first lower left molar   First and third lower right molars   Third lower right and left molars   ND
  Synkinesis                                                                                     Bimanual                                                                Bipodalic                            Bimanual                            ND
  Short fourth metacarpal                                                                        Right/left                                                              No                                   No                                  ND

**Notes:**

G, external genitalia; P, pubic hair; B, breast development;

C-R, right cryptorchidism, C-L, left cryptorchidism.

**Abbreviation:** ND, no data available.