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 {#s1}
============

Homozygous truncating mutations in the basic helix-loop-helix transcription factor *PTF1A* cause pancreatic agenesis and very severe neurodevelopmental problems, including central hypoventilation and total cerebellar agenesis ([@B1]--[@B3]), and all six affected individuals from four families showed a high degree of phenotypic concordance and none survived for more than 4 months.

A combined linkage, genome sequencing, and epigenomic annotation strategy recently identified homozygous mutations in a novel enhancer located 25 kb downstream from the *PTF1A* gene ([@B4]). Patients with biallelic mutations in this enhancer have isolated pancreatic agenesis without cerebellar involvement. Sparing of the cerebellum suggests that the enhancer is tissue specific to the pancreas.

A mouse model relating *Ptf1a* dosage to pancreatic phenotype ([@B5]) resulted in pancreatic hypoplasia and glucose intolerance in a dosage-dependent manner. The pancreatic phenotype consisted of reduced pancreatic bud size, misspecification of pancreatic progenitors, reduced branching morphogenesis of the exocrine pancreas, and a reduction in the ratio of β-cells to non--β-cells in pancreatic islets. In this model system, *Ptf1a* RNA levels were correlated with the endocrine and exocrine pancreatic phenotype.

Biallelic mutations in a second transcription factor gene, *PDX1,* also result in nonsyndromic pancreatic agenesis and have been identified in three unrelated cases ([@B6]--[@B8]). Hypomorphic mutations result in neonatal diabetes in the absence of exocrine pancreatic insufficiency ([@B9],[@B10]). A hypomorphic mutation in the transcription factor gene *Pdx1* in mice has also been modeled ([@B11]) and been shown to play a role in the transition from pancreatic progenitor to endocrine progenitor, with a reduction in the number of endocrine lineages.

To date, no *PTF1A* coding hypomorphic mutations have been identified. We now report four individuals from two separate sibships in each of whom the same novel *PTF1A* coding mutation, p.P191T, was identified. All four individuals were diagnosed with neonatal diabetes, but cerebellar pathology was absent. We performed functional studies on this mutation to investigate our hypothesis that p.P191T is a hypomorphic *PTF1A* mutation.

Research Design and Methods {#s2}
===========================

Genetics {#s3}
--------

### Homozygosity Mapping {#s4}

Homozygosity mapping was carried out as described previously ([@B12]) in one patient with pancreatic agenesis of unknown etiology and their sibling with neonatal diabetes. Pancreatic agenesis was defined as neonatal diabetes requiring insulin treatment and exocrine pancreatic insufficiency defined as requiring enzyme replacement therapy ([@B13]).

### Next-Generation Sequencing Assay {#s5}

A targeted next-generation sequencing assay was used to sequence *PTF1A* and 22 other genes in which mutations have been reported to cause neonatal diabetes ([@B14]). We sequenced DNA samples from 259 probands with neonatal diabetes diagnosed before 6 months and no known genetic etiology. Nine of these patients had pancreatic agenesis. Mutations in *ABCC8, KCNJ11*, and *INS* had previously been excluded by Sanger sequencing. We had also excluded *EIF2AK3* mutations in patients born to consanguineous parents.

### Sanger Sequencing {#s6}

Mutations were confirmed by PCR/Sanger sequencing and tested in other relatives.

Functional Work {#s7}
---------------

### Cell Transfections {#s8}

The reporter plasmids Ela1p.luc, with a minimal promoter directing the luciferase gene of pGL3 basic, and 3Rbpjl.Ela1p.luc, with three tandem repeats of the proximal PTF1 binding site of the *Rbpj1* gene upstream of Ela1p.luc, have been described ([@B15]). The p.P191T mutation was introduced into the human PTF1A coding sequence by site-directed mutagenesis as previously described ([@B16]). DNA was introduced into the human embryonic kidney (HEK) 293 cell line (CRL-1573; American Type Culture Collection) with FuGene 6 (Roche, Indianapolis, IN), according to the manufacturer's instructions. All transfections were normalized based on the β-galactosidase activity of a cotransfected reporter plasmid, pCMVbeta (Clontech, Mountain View, CA).

### Electrophoretic Mobility Shift Assays {#s9}

The subunits of PTF1 were synthesized by in vitro transcription and translation using a TnT Coupled Reticulocyte Lysate System (Promega, Madison, WI). The wild-type and p.P191T mutant PTF1A plasmids described above were also used for in vitro protein synthesis. The plasmid bearing human RBPJ has previously been described ([@B16]). The expression plasmid bearing a partial human E12/TCF3 cDNA in pCITE2a was a gift from Eric Olson, of The University of Texas Southwestern Medical Center, and was originally derived from E12R ([@B20]). The products of in vitro transcription and translation were quantified by \[^35^S\]methionine incorporation and adjustment according to the number of methionine residues in each protein.

Electrophoretic mobility shift assays (EMSAs) were performed with the method of Sawada and Littman ([@B17]) with slight modifications. The double-stranded oligonucleotide probe encompassed the proximal PTF1 binding site of the mouse *Rbpjl* gene ([@B15]) and was 5′-end labeled with ^32^P. The sequence of the top strand is GACACCTGCTGGGCAGATGTAGGCTTCCCACGG. ImageQuant software was used to analyze Phosphorimager scans of the EMSA gels (GE Healthcare, Life Sciences, Pittsburgh, PA).

Results {#s10}
=======

Homozygosity analysis of genome-wide single nucleotide polymorphisms identified a homozygous region (31 Mb) on chromosome 10 that was shared between two affected siblings born to consanguineous parents. Sanger sequencing of *PTF1A* identified a novel homozygous missense mutation, p.P191T (c.571C\>A; p.Pro191Thr), in both siblings. Analysis of *PTF1A* by targeted next-generation sequencing of nine patients with pancreatic agenesis identified one additional proband with the p.P191T mutation. This patient's affected sibling was also homozygous for p.P191T. No additional *PTF1A* mutations were identified in a further 259 patients with neonatal diabetes.

P191T Mutation Characteristics {#s11}
------------------------------

The p.P191 residue is located in the highly conserved helix-loop-helix domain that is critical for dimerization and DNA binding ([Fig. 1](#F1){ref-type="fig"}). The p.P191T missense variant is not present in any public variant databases (1000 Genomes, NHLBI Exome Sequencing Project Exome Variant Server, or ExAC Browser). In silico analysis by SIFT and PolyPhen predicts the variant to be pathogenic. All four affected patients were homozygous for the p.P191T mutation and their parents were heterozygous carriers. The patients are from Saudi Arabia and Kuwait, raising the possibility of a founder mutation. Analysis of microsatellite markers flanking *PTF1A* showed a shared haplotype of 8 Mb (data not shown), consistent with a founder mutation segregating in the two families.

![*A*: Schematic representation of the PTF1A protein and location of the mutations identified in all previously reported cases ([@B1]--[@B3]) and the p.P191T mutation found in the four current cases. *B*: Amino acid conservation in the bHLH domain. The p.P191T is the only missense mutation identified to date. It is located at a conserved residue within the basic helix-loop-helix (bHLH) domain (highlighted). ¥The p.Ala146_Arg154delinsGly mutation was previously reported as c.437_460del, p.Ala146_Arg154delfsX115 ([@B3]).](db151666f1){#F1}

Clinical Characteristics of Patients With Homozygous P191T Mutation {#s12}
-------------------------------------------------------------------

The male proband in family I (I-1) was the first child born to first cousins of Arabic descent with a birth weight of 1.98 kg at 38 weeks' gestation. He was noted to be hyperglycemic (350 mg/dL) on the first day of life while on a glucose infusion, which was discontinued and an insulin infusion commenced for 20 h. On day 6, when on no treatment, his plasma glucose remained high (240 mg/dL) with low insulin of 0.1 μU/L (normal range 1.4--14) and C-peptide of 0.1 ng/mL (normal range 0.9--4.3), confirming a diagnosis of neonatal diabetes. Insulin was recommended at 2 months of age as most of his glucose values were 200--300 mg/dL and his HbA~1c~ was 7%. The treatment has continued, with HbA~1c~ 8.1%. The baby continued to have poor weight gain despite recommencing insulin and steatorrhea was observed, suggesting pancreatic insufficiency, which was confirmed by elevated fecal fat, elevated stool chymotrypsin, and low serum trypsinogen ([Table 1](#T1){ref-type="table"}). Exocrine pancreatic replacement therapy was initiated at 3 months of age. Other clinical features were a small patent ductus arteriosus and a small atrial septal defect, which were detected by echo after a cardiac murmur was noted; these did not require any intervention.

###### 

Clinical characteristics of patients studied

                                                                I-1                                                                                                                                 I-2                                                                                                    II-1                                 II-2
  ------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------ ------------------------------------ ----------------------------------------------------------
  Present age                                                   12 years                                                                                                                            9 years                                                                                                Deceased at 12 weeks due to sepsis   2 years
  Sex                                                           Male                                                                                                                                Female                                                                                                 Male                                 Female
  Country of origin                                             Saudi Arabia                                                                                                                        Saudi Arabia                                                                                           Kuwait                               Kuwait
  Birth weight (g)                                              1,980                                                                                                                               2,000                                                                                                  1,275                                1,400
  Percentile                                                    0.4                                                                                                                                 2                                                                                                      0.1                                  \<0.1
  Gestational age                                               38 weeks                                                                                                                            37 weeks                                                                                               34 weeks                             36 weeks
  Age at diabetes diagnosis/ age of permanent insulin therapy   1 day/2 months                                                                                                                      1 day/4 months                                                                                         1 day/1 day                          8 days/8 days
  Diabetes treatment                                            Insulin                                                                                                                             Insulin                                                                                                Insulin                              Insulin
  Exocrine pancreatic insufficiency requiring replacement?      Yes                                                                                                                                 No                                                                                                     Yes                                  Yes
  Clinical basis of exocrine insufficiency                      Steatorrhea, failure to thrive                                                                                                      No symptoms of exocrine insufficiency                                                                  Failure to thrive                    Steatorrhea, failure to thrive
  Biochemical basis of exocrine insufficiency                   Fecal fat: 3 g/24 h stool Stool chymotrypsin: 1 U/g (normal range 4--10 U/g) Serum trypsinogen: 4 μg/L (normal range 15--25 μg/L)   Fecal fat: 0.5 g/24 h Stool chymotrypsin: 5 U/g (4--10 U/g) Serum trypsinogen: 18 μg/L (15--25 μg/L)                                        Fecal elastase: undetectable (\>200 μg elastase/g stool)
  Exocrine pancreatic replacement regimen                       10,000 U/day of lipase                                                                                                              None                                                                                                                                        15,000 U/day of lipase
  Age at initiation of exocrine pancreatic replacement          12 weeks                                                                                                                            Not applicable                                                                                         Not applicable                       10 weeks
  Clinical neurocognitive function                              Normal neurocognitive development with some disruption of eye movements on tracking                                                 Normal neurocognitive development                                                                                                           Normal neurocognitive development
  Brain imaging                                                 Not performed                                                                                                                       Not performed                                                                                          Not performed                        Normal (MRI)

His younger sister (I-2) was born at 37 weeks' gestation with a birth weight 2.00 kg. Diabetes was diagnosed on the first day of life with a glucose of 300 mg/dL and and she was treated with insulin for 4 days. Insulin was commenced at 4 months due to consistently high blood glucose values (\>200 mg/dL) and HbA~1c~ of 7%; the treatment has continued with HbA~1c~ 7--8%. Exocrine pancreatic supplementation has not been required and biochemical parameters (fecal fat, stool chymotrypsin, and serum trypsinogen) are within normal limits, albeit at the lower limit of the normal range ([Table 1](#T1){ref-type="table"}).

Ultrasound scanning of the abdomen of the two siblings in family I was performed in the first week of life and showed normal liver and contracted gallbladder but failed to identify the pancreas. Parents declined further imaging of the abdomen.

Both siblings in family I underwent detailed neurodevelopmental assessment by a neurodevelopmental pediatrician at the age of 12 years in the older and at 9 years in the younger sibling. Evaluation revealed normal neurological examination apart from searching eye movements with horizontal nystagmus in patient I-1. There are no other clinical abnormalities referable to cerebellar function. Parents declined to perform brain MRIs. The rest of the clinical neurodevelopmental assessment was age-appropriate, including vision, hearing, expressive and receptive language, gross motor and fine motor skills, social skills, and school performance. Their clinical neurodevelopmental assessment is similar to their unaffected siblings. However, formal neuropsychological testing was not performed.

The male proband in family II (II-1) was the first child born to consanguineous parents of Arabic descent. Birth weight was 1.275 kg at 34 weeks' gestation. Neonatal diabetes was diagnosed on the first day of life and treated with insulin. There was severe failure to thrive gaining only 250 g in the first 12 weeks of life, but biochemical tests for pancreatic malabsorption were not performed. The patient died of necrotizing enterocolitis and overwhelming sepsis at the age of 12 weeks. No further clinical details are available.

His younger sibling is a female child (II-2) born at 36 weeks' gestation with a birth weight 1.40 kg. Neonatal diabetes was diagnosed at the age of 8 days and treated with insulin. She failed to thrive and fecal elastase was undetectable, confirming a diagnosis of exocrine pancreatic insufficiency. Exocrine replacement therapy was initiated at 10 weeks of age. Neurocognitive development and neurological examination are normal at the age of 2 years. Brain and cerebellar MRI scan was normal at 3 months of age.

Functional Work {#s13}
---------------

Transfection of the p.P191T mutation into HEK293 cells resulted in a 75% reduction in transcriptional activity compared with the wild-type protein ([Fig. 2](#F2){ref-type="fig"}). A range in the amount of expression plasmids was tested to ensure that the activities of the wild-type plasmids were proportional to the plasmid quantity and, thus, not saturated in the assay. The mean ± SD activity of the mutant PTF1A at three different concentrations within the linear range was 25 ± 4.9% of the activity of the wild-type protein (*P* \< 0.001) ([Fig. 2](#F2){ref-type="fig"}).

![The activity of human PTF1A-P191T in transfected cells. The reporter plasmid 3Rbpjl.Ela1p.luc was cotransfected with expression plasmids for wild-type (w.t.) PTF1A, PTF1A-P191T, or an insertless vector into HEK293 cells. Three different amounts of each expression plasmid were used to determine whether response was proportional to plasmid quantity and, therefore, that the proteins produced were not in excess. Four independent transfections were analyzed for 10 and 5 ng and two for 2.5 ng. Error bars are SD. The asterisks indicate *P* values \<0.05 for each pairwise comparison.](db151666f2){#F2}

To determine the DNA binding characteristics of human PTF1A-P191T relative to wild type, EMSAs were performed with an *Rbpjl* proximal PTF1 binding site as the radiolabeled probe ([Fig. 3](#F3){ref-type="fig"}). For the PTF1A:E12 dimer, the binding of the p.P191T mutant was 34 ± 7% of wild type (*n* = 3), whereas for the PTF1A:E12:RBPJ trimer, the binding of the p.P191T mutant was 88 ± 19% of wild type (*n* = 4).

![The DNA binding of wild-type PTF1A and PTF1A-P191T as part of the PTF1A:E12 dimer or PTF1A:E12:RBPJ trimer in EMSA. The relative abilities of wild-type and p.P191T mutant PTF1A to form complexes with E12 and RBPJ on the PTF1 binding site of the *Rbpj1* gene was tested in vitro. Lanes 3a and 4a show lanes 3 and 4 with an expanded grayscale such that the PTF1 trimeric complex bands are not saturated. PTF1A alone does not bind DNA. 2, PTF1A:E12 dimer; 3, PTF1A:E12:RBPJ trimer; d, E12 homodimer; J, RBPJ monomer; M, p.P191T mutant; W, PTF1A wild type.](db151666f3){#F3}

Discussion {#s14}
==========

We report a novel hypomorphic homozygous *PTF1A* mutation in four affected individuals from two families who are likely to have inherited the mutation from a common distant ancestor. The clinical phenotype is of neonatal diabetes with reduced exocrine function and normal neurological function and appearance. This clinical phenotype is distinct from the previously reported syndrome caused by homozygous *PTF1A* truncating mutations where all six patients (from four families) died in the first 4 months of life ([@B1]--[@B3]) of severe neurological complications and had pancreatic and cerebellar agenesis. In vitro functional studies are consistent with the p.P191T missense mutation being a hypomorphic mutation.

The main clinical feature of homozygosity for the p.P191T missense mutation is pancreatic aplasia/hypoplasia. All four patients had neonatal diabetes and two of the four have clinical and biochemical evidence of clinically significant exocrine dysfunction of the pancreas requiring replacement therapy. The third patient had clinical features suggestive of exocrine insufficiency but died of sepsis at the age of 12 weeks, and the fourth patient had diabetes requiring insulin treatment but no clinical features of exocrine failure, albeit with pancreatic enzyme levels close to the lower limit of the normal range.

All four patients had normal neurological development and function. This is in contrast to the patients with homozygous null *PTF1A* mutations who had very severe neurological developmental problems, central hypoventilation, and total cerebellar agenesis. One patient had some disrupted eye movements, but the phenotype was not consistent with significant cerebellar dysfunction. A normal cerebellum was seen in the patient who had brain imaging.

The moderate phenotypic variability seen in these four patients is also seen in patients who have other pancreatic transcription factor mutations affecting the pancreatic stem cell. Different phenotypes due to the same mutation are seen in patients with mutations in *GATA6* ([@B18]) and *HNF1B* ([@B19]). Phenotypic variability is also seen in patients with mutations in the homeodomain transcription factor *PDX1*: biallelic mutations in this gene have been reported in patients with pancreatic agenesis ([@B6]--[@B8]), neonatal diabetes with biochemical but not clinical evidence of exocrine insufficiency ([@B9],[@B10]), and neonatal diabetes with normal exocrine function, both clinically and biochemically ([@B10]).

The p.P191T missense mutation is located in the highly conserved helix-loop-helix domain of *PTF1A*, a region that is critical for dimerization with a common basic helix-loop-helix E-protein, such as E12/E47, and DNA binding. Proline 191 terminates helix 1, and substitution of threonine at that position could favor the extension of the first α-helix almost a full turn and thereby shorten the loop region, misposition the second α-helix, and make binding to the E-protein partner less favorable. Indeed, in vitro functional studies demonstrated an effect on DNA binding and that transactivation activity was reduced by 75% compared with the wild-type protein. This residual activity is consistent with p.P191T being a hypomorphic mutation.

Reduced *Ptf1a* dosage in a mouse model ([@B5]) resulted in pancreatic hypoplasia and glucose intolerance in a dosage-dependent manner. In hypomorphic mutant mice, pancreatic bud size was small and substantial proportions of pancreatic progenitors were misspecified to the common bile duct and duodenal cells. Exocrine pancreatic branching morphogenesis was reduced, and there was a reduction in the ratio of β-cells to non--β-cells in pancreatic islets. In this model system, *Ptf1a* RNA levels could be correlated with the endocrine and exocrine pancreatic phenotype. We have clearly shown a similar correlation between mutation severity and phenotype in humans.

We conclude that hypomorphic *PTF1A* missense mutations can cause isolated pancreatic agenesis or neonatal diabetes. Hypomorphic mutations in this gene should be considered in patients presenting with diabetes in the neonatal period.

**Acknowledgments.** The authors thank the families for participating in this study. They are grateful to Annet Damhuis and Anna-Maria Bussell and the Research Center, College of Medicine, King Saud University for their technical assistance and to Ward Coats (Department of Molecular Biology, The University of Texas Southwestern Medical Center) for helpful discussions regarding PTF1A structure.

**Funding.** G.H.S. and R.J.M. were supported by a National Institutes of Health grant R01-DK061220. A.T.H. and S.E. are the recipients of a Wellcome Trust Senior Investigator award, which funded the genetic/clinical part of this study (WT098395). A.T.H. is employed as a core member of staff within the National Institute for Health Research (NIHR)-funded Exeter Clinical Research Facility and is an NIHR senior investigator.

**Duality of Interest.** No potential conflicts of interest relevant to this article were reported.

**Author Contributions.** J.A.L.H., S.E.F., E.d.F., R.C., and S.E. designed, performed, and interpreted the genetic studies. C.S.-S., K.H., S.M., M.A., and A.T.H. designed and performed the clinical studies. G.H.S. and R.J.M. designed, performed, and interpreted the functional studies. J.A.L.H. and G.H.S. wrote the first draft of the manuscript with input from A.T.H., R.J.M., and S.E. All authors reviewed and commented on the draft manuscript and reviewed the submitted manuscript. S.E. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

[^1]: J.A.L.H., G.H.S., and C.S.-S. contributed equally to this study.