Gene: 1673	Name: Ptch2	Family: Sterol Sensing Domain	Subfamily: Novel	Accession: NM_008958	GI: 6679516

Gene 1673
Summary of Phenotypic Analysis

Changes related to genotype:

• Necropsy:
• Spleen, size increased, 49 day homozygous male.
• Spleen weight and spleen weight/body weight ratio, high, 49 day homozygous males.
• Histopathology:
• Spleen, extramedullary hematopoiesis, increased , 49 day homozygous males.
• Bone marrow, myeloid:erythroid ratio, increased, 49 day homozygous males
• Hematology:
• Anemia, 49 day homozygous males.
• Hemoglobin, hematocrit, and RBC count, low.
• RBC morphological abnormalities.
• Neutrophil percentage, high, 49 day homozygous males.

ES cells derived from the 129/OlaHsd mouse substrain were used to generate chimeric mice. F1 mice were generated by breeding with C57BL/6 females. F2 homozygous mutant mice were produced by intercrossing F1 heterozygous males and females.

Wild-type control mice and homozygous mutant mice were evaluated by the following examinations or tests:

• Physical examination
• Necropsy, including body length, body weight, and organ weight measurements
• Histological examination of tissues and organs
• Bone marrow section evaluation
• Complete blood counts and differential cell counts
• Clinical chemistry panels
• Densitometry
• Behavioral tests

Necropsy
Spleen weight and spleen weight/body weight ratio were high in two 49 day cohort homozygous mutant males that correlated with increased spleen size in one of them.

Histopatholgy
There was moderate to severe diffuse increased extramedullary hematopoiesis of the spleen in two 49 day cohort homozygous mutant males. Myeloid:erythroid ratio was slightly high in the bone marrow of all 49 day homozygous mutant males.

Hematology
When compared to age- and gender-matched wild-type historical control mice, there were findings consistent with a mild anemia including hemoglobin, hematocrit and RBC count that were slightly low in two 49 day cohort homozygous mutant males. MCV was slightly high in one homozygous mutant male. Lastly, there were mild RBC morphological abnormalities on the peripheral blood smear, including anisocytosis, poikilocytosis, and polychromasia. The percentage of neutrophils was mildly to moderately high in both male 49 day cohort homozygous mutant males.

Gene 1673
Behavior

There were no significant differences detected in the homozygous mutant animals when compared with age- and gender-matched wild-type control mice.

Homozygous mutant and wild-type control mice were evaluated for phenotypic changes by testing on seven behavioral tasks: Open field test, Tail suspension test, Rotarod test, Startle response/PPI test, Tail flick test, Hot plate test, and Metrazol test.

Mouse ID numbers are as follows for the N1 generation:
10 homozygous mutant males (414628, 414632, 416832, 421997, 422000, 422001, 434477, 434480, 440590, 443446)
37 wild-type control males (403896, 409605, 412165, 414290, 414598, 414627, 415033, 415462, 415724, 417034, 417598, 417825, 418337, 418343, 418831, 418969, 419144, 420261, 420755, 421432, 422002, 423001, 425724, 431728, 431731, 431789, 431795, 437527, 437528, 438688, 439643, 440265, 440266, 440591, 441679, 443806, 444608)

ES cells derived from the 129/OlaHsd mouse substrain were used to generate chimeric mice.  F1 mice were generated by breeding with C57BL/6 females. The resultant F1N0 heterozygotes were backcrossed to C57BL/6 mice to generate F1N1 heterozygotes.  F2N1 homozygous mutant mice were produced by intercrossing F1N1 heterozygous males and females.

Behavior Findings:
There were no genotype-related or biologically significant differences noted between homozygous mutant and wild-type control mice for any of the parameters evaluated during behavior testing.

Gene 1673
Expression Summary

Taqman Summary:
RNA transcripts are detectable in all tissues analyzed.

The highest levels of RNA transcripts are detectable in testis.

Moderate levels of RNA transcripts are detectable in skin, urinary bladder, pituitary gland, adrenal gland, epididymis, coagulating gland and white fat.
   
Lower levels of RNA transcripts are also detectable in all other tissues analyzed: whole brain, cortex, subcortical region, cerebellum, brainstem, olfactory bulb, spinal cord, eye, Harderian glands, heart, lung, liver, pancreas, kidney, spleen, thymus, lymph nodes, bone marrow, gallbladder, salivary gland, skeletal muscle, tongue, stomach, small intestine, large intestine, cecum, seminal vesicle, prostate gland, ovary and uterus.

LacZ Summary:
LacZ expression was detected in several of the organs examined.  Striking staining was observed in hair follicles, pars distalis of the pituitary gland, wholemount brain and the purkinje cell layer on coronal sections of cerebellum.  Staining was observed in different tissue and cell types, including epithelium, neurons, connective tissue and a few blood vessels.  Staining was also observed in cells adjacent to blood vessels.

LacZ expression was detected in:  brain, spinal cord, sciatic nerve, aorta, heart, lung, liver, pancreas, kidney, urinary bladder, thyroid gland, trachea, pituitary gland, adrenal glands, skeletal muscle, skin, testis, prostate gland, ovary, uterus and cervix.

LacZ expression was not detected in:  eyes, thymus, spleen, lymph nodes and bone marrow.

Gene 1673
Densitometry

There were no significant differences detected in the homozygous mutant mice when compared with age- and gender-matched wild-type historical control mice.

The following mice were evaluated by dual-energy x-ray absorptiometry.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (359950, 359951, 372010)
3 homozygous mutant males (359943, 359945, 359946)
3 heterozygous mutant females (372013, 372015, 379293)
3 heterozygous mutant males (372004, 372005, 372006)
2 wild-type control females (368310, 372014)
2 wild-type control males (372003, 372007)

Bone Mineral Density (BMD in g/cm² ), fat % (fat percentage expressed as a percentage of body soft tissue compartment), and R-value of soft tissue were calculated from Bone Mineral Content (BMC in g), bone and tissue areas (cm² ), and total tissue mass (g) generated by a PIXImus densitometer.

Densitometric Findings:

Incidental densitometric differences were present between some mice. These findings were considered to represent background differences occasionally seen in this strain of mice, differences due to spontaneous disease, age-related differences, and/or differences of a nonspecific etiology. They were not considered to be genotype related.

Gene 1673
Histopathology

Changes related to genotype: 

• Spleen, extramedullary hematopoiesis, increased , 49 day homozygous males.
• Bone marrow, high myeloid:erythroid ratio, 49 day homozygous males.

Tissues from the following mice were evaluated histologically.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (359950, 359951, 372010)
3 homozygous mutant males (359943, 359945, 359946)
3 heterozygous mutant females (372013, 372015, 379293)
3 heterozygous mutant males (372004, 372005, 372006)
2 wild-type control females (368310, 372014)
2 wild-type control males (372003, 372007)

Histopathology Findings:

There was moderate to severe diffuse increased extramedullary hematopoiesis of the spleen in two 49 day cohort homozygous mutant males (359943, 359945). This finding correlated with increased spleen size, and high spleen weight and spleen weight/body weight ratio at physical examination, and mild anemia at hematological examination. Additional mice are pending.

Bone marrow was examined in sections of sternum, vertebrae, and/or femur and tibia. Marrow cellularity, myeloid:erythroid (M:E) ratio, myeloid and erythroid maturation sequences, and numbers of megakaryocytes were evaluated. The myeloid:erythroid ratio was slightly high in all 49 day homozygous mutant males. This correlated with the high percentage of neutrophils at hematological examination.

No Significant Abnormalities:

The following tissues were examined and considered to have no genotype-related abnormality: brain, pituitary gland, ears, nasal cavity, salivary glands, oral cavity, lymph nodes, aorta, lungs, liver, gallbladder, pancreas, kidneys, adrenal glands, urinary bladder, stomach, small and large intestines, larynx, esophagus, trachea, thyroid gland, thymus, tongue, skeletal muscle, sciatic nerve, mammary glands, vertebrae, spinal cord, bone (skull, sternum, femur, tibia, and stifle joint), reproductive tract (including gonads), eyes, Harderian glands, and integumentary system (skin and either clitoral or preputial glands).

Incidental lesions were present in some tissues. These findings were considered to represent background lesions occasionally seen in this strain of mice, lesions due to spontaneous disease, age-related lesions, and/or lesions of a nonspecific etiology. They were not considered to be genotype related.

Gene 1673
Necropsy 

Changes related to genotype:

• Spleen, size increased, 49 day homozygous male.
• Spleen weight and spleen weight/body weight ratio, high, 49 day homozygous males.

The following mice were necropsied. Body weight, body length, and organ weights were obtained, and gross pathological findings were recorded.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (359950, 359951, 372010)
3 homozygous mutant males (359943, 359945, 359946)
3 heterozygous mutant females (372013, 372015, 379293)
3 heterozygous mutant males (372004, 372005, 372006)
2 wild-type control females (368310, 372014)
2 wild-type control males (372003, 372007)

Mice were examined for the following observables: adrenal glands, body length, body weight, bone marrow, bone - cranium, bone - femur, bone - sternum, bone - stifle joint, bone - vertebral column, brain, cecum, colon, duodenum, epididymis - seminal vesicle, esophagus, eyes, gallbladder, general appearance, Harderian glands, heart, heart weight, ileum, jejunum, kidney weight, kidneys, liver, liver weight, lungs, lymph nodes, mesentery, ovaries, pancreas, penis, salivary glands, sciatic nerve, scrotum, skeletal muscle, skin, skinned mouse, spleen, spleen weight, stomach, testes, testes - epididymis weight, thymus, thymus weight, tongue, trachea, urinary bladder, urine, uterus, and vagina. (Gender-specific observables apply to the appropriate gender.)

Necropsy Findings:

Spleen size was increased in spleen size in one 49 day cohort homozygous mutant male (see below; 349945).

There were no other genotype-related or biologically significant differences noted between mutant and wild-type control mice for any of the parameters evaluated at necropsy. Incidental lesions were present in some tissues. These findings were considered to represent background lesions occasionally seen in this strain of mice, lesions due to spontaneous disease, age-related lesions, and/or lesions of a nonspecific etiology. They were not considered to be related to genotype.

Body and Organ Weight Findings:

Spleen weight and spleen weight/body weight ratio were high in two 49 day cohort homozygous mutant males (349943, 349945) and these findings correlated with increased spleen size in one of them (349945).  It also correlated with moderate to severe extramedullary hematopoiesis at histopathological examination and mild anemia at hematological examination. Additional mice are pending.

Other differences in body length, body weight, organ weights, and/or organ weight to body weight ratios were present between individual mice. The variability between mice usually fell within our historical reference ranges and was not correlated with genotype.

Gene 1673
Clinical Chemistry

There were no significant differences in the homozygous mutant mice when compared with age- and gender-matched historical wild-type control mice.

Serum samples from the following mice were evaluated by a clinical chemistry panel.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (359950, 359951, 372010)
3 homozygous mutant males (359943, 359945, 368314)
3 heterozygous mutant females (372013, 379283, 379293)
3 heterozygous mutant males (372004, 372006, 372009)
2 wild-type control females (368310, 372016)
2 wild-type control males (372003, 372007)

Values for the various analytes evaluated were generally similar between homozygous mutant and historical wild-type control mice. Although variations in clinical chemistry values were present in some mice, they were not related to genotype and, thus, were not considered phenotypically relevant.

Gene 1673
Hematology

Changes related to genotype:

• Anemia, 49 day homozygous males.
• Hemoglobin, hematocrit, and RBC count, low.
• RBC morphological abnormalities.
• Neutrophil percentage, high, 49 day homozygous males.

Blood samples from the following mice were evaluated by a complete blood count and differential cell count.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (359951, 372010, 372017)
3 homozygous mutant males (359943, 359945, 368317)
3 heterozygous mutant females (372015, 379285, 379293)
3 heterozygous mutant males (372004, 372005, 372006)
2 wild-type control females (368310, 372014)
2 wild-type control males (372003, 372007)

When compared to age- and gender-matched wild-type historical control mice, hemoglobin, hematocrit and RBC count were slightly low in two 49 day cohort homozygous mutant males (359943, 359945). MCV was slightly high in one homozygous mutant male (359945). There were mild RBC morphological abnormalities on the peripheral blood smear, including anisocytosis, poikilocytosis, and polychromasia. These findings were consistent with a mild anemia. Additional mice are pending.

The percentage of neutrophils was mildly to moderately increased in both male 49 day cohort homozygous mutant males. This finding correlated with a slightly high myeloid:erythroid ratio at histopathological examination of the bone marrow.

Although minor variations of other hematological values were present in some mice, these changes were not related to genotype and, thus, were not considered phenotypically relevant.

Gene 1673
Physical Examination

There were no significant differences detected in the homozygous mutant mice when compared with age- and gender-matched wild-type historical control mice.

The following mice were evaluated by physical examination.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (359950, 359951, 372010)
3 homozygous mutant males (359943, 359945, 359946)
3 heterozygous mutant females (372013, 372015, 379293)
3 heterozygous mutant males (372004, 372005, 372006)
2 wild-type control females (368310, 372014)
2 wild-type control males (372003, 372007)

Mice were examined in detail as follows: anus, behavior, body shape, claws, coat - fur, coat color - back, coat color - belly, ear - left, ear - right, eye - left, eye - right, eye color - left, eye color - right, feces, forelimb - left, forelimb - right, forelimb number of amputated digits - left, forelimb number of amputated digits - right, forelimb number of digits - left, forelimb number of digits - right, general appearance, genitals - female, genitals - male, hair type, head shape, hindlimb - left, hindlimb - right, hindlimb number of amputated digits - left, hindlimb number of amputated digits - right, hindlimb number of digits - left, hindlimb number of digits - right, injuries, lesions, limb shape, locomotor, lumps - masses, mammary glands, mice in cage, respiration, skin appearance, snout, swelling - joints, tail, teeth color, teeth length, urine, and whiskers. (Gender-specific observables apply to the appropriate gender.)

Individual homozygous mutant mice had only occasional minor differences in observed physical features compared to wild-type control mice. These findings were considered to represent individual variability, background features occasionally seen in this strain of mice, findings due to spontaneous disease, age-related findings, and/or findings of a nonspecific etiology. However, none of these differences were regarded as biologically significant or genotype related.